Method of detecting DNA encoding a β-glucosidase from a filamentous fungus

ABSTRACT

A process for expressing extracellular β-glucosidase in a filamentous fungus by expressing a fungal DNA sequence encoding enhanced, deleted or altered β-glucosidase in a recombinant host microorganism is disclosed. Recombinant fungal cellulase compositions containing enhanced, deleted or altered expression of β-glucosidase is also disclosed.

This is a Divisonal of U.S. Ser. No. 08/248,586 filed May 24, 1994, nowabandoned, which is a continuation application of 07/807,028 filed Dec.10, 1991, now abandoned, which is a continuation-in-part of 07/625,140filed Dec. 10, 1990, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to cellulase preparations and compositionshaving increased or decreased cellulolytic capacity. The inventionfurther relates to a nucleotide sequence of the bg11 gene encodingextracellular β-glucosidase from a filamentous fungi, a plasmid vectorcontaining the gene encoding extracellular β-glucosidase andtransformant strains with increased copy numbers of the β-glucosidase(bg11) gene introduced into the genome. More particularly, the presentinvention relates to Trichoderma reesei strains that have increased orno levels of expression of the bg11 gene resulting in enhanced or noextracellular β-glucosidase protein levels that can be used inconjunction with other compositions to produce a cellulase producthaving increased or decreased cellulolytic capacity.

2. State of the Art

Cellulases are known in the art as enzymes that hydrolyze cellulose(β-1,4-glucan linkages), thereby resulting in the formation of glucose,cellobiose, cellooligosaccharides, and the like. As noted by Wood etal., "Methods in Enzymology", 160, 25, pages 234 et seq. (1988) andelsewhere, cellulase produced by a given microorganism is comprised ofseveral different enzyme classes including those identified asexocello-biohydrolases (EC 3.2.1.91) ("CBH"), endoglucanases (EC3.2.1.4) ("EG"), β-glucosidases (EC 3.2.1.21) ("BG"). Moreover, thefungal classifications of CBH, EG and BG can be further expanded toinclude multiple components within each classification. For example,multiple CBHs and EGs have been isolated from a variety of bacterial andfungal sources including Trichoderma reesei which contains 2 CBHs, i.e.,CBH I and CBH II, and at least 3 EGs, i.e., EG I, EG II, and EG IIIcomponents.

The complete cellulase system comprising components from each of theCBH, EG, and BG classifications is required to efficiently convertcrystalline forms of cellulose to glucose. Isolated components are farless effective, if at all, in hydrolyzing crystalline cellulose.Moreover, a synergistic relationship is observed between the cellulasecomponents particularly if they are of different classifications. Thatis to say, the effectiveness of the complete cellulase system issignificantly greater than the sum of the contributions from theisolated components of the same classification. In this regard, it isknown in the art that the EG components and CBH components synergistic-ally interact to more efficiently degrade cellulose. See, for example,Wood, Biochem. Soc. Trans., 13, pp. 407-410 (1985).

The substrate specificity and mode of action of the different cellulasecomponents varies with classification, which may account for the synergyof the combined components. For example, the current accepted mode ofcellulase action is that endoglucanase components hydrolyze internalβ-1,4-glucosidic bonds, particularly, in regions of low crystallinity ofthe cellulose and exo-cellobiohydrolase components hydrolyze cellobiosefrom the non-reducing end of cellulose. The action of endoglucanasecomponents greatly facilitates the action of exo-cellobiohydrolases bycreating new chain ends which are recognized by exo-cellobiohydrolasecomponents.

β-Glucosidases are essential components of the cellulase system and areimportant in the complete enzymatic breakdown of cellulose to glucose.The β-glucosidase enzymes can catalyze the hydrolysis of alkyl and/oraryl β-D-glucosides such as methyl β-D-glucoside and p-nitrophenylglucoside, as well as glycosides containing only carbohydrate residues,such as cellobiose. The catalysis of cellobiose by β-glucosidase isimportant because it produces glucose for the microorganism and furtherbecause the accumulation of cellobiose inhibits cellobiohydrolases andendoglucanases thus reducing the rate of hydrolysis of cellulose toglucose.

Since β-glucosidases can catalyze the hydrolysis of a number ofdifferent substrates, the use of this enzyme in a variety of differentapplications is possible. For instance, some β-glucosidases can be usedto liberate aroma in fruit by catalyzing various glucosides presenttherein. Similarly, some β-glucosidases can hydrolyze grape monoterpenylβ-glucosidase which upon hydrolysis, represents an important potentialsource of aroma to wine as described by Gunata et al, "Hydrolysis ofGrape Monoterpenyl β-D-Glucosides by Various β-Glucosidases", J. Agric.Food Chem., Vol. 38, pp. 1232-1236 (1990).

Furthermore, cellulases can be used in conjunction with yeasts todegrade biomass to ethanol wherein the cellulose degrades cellobiose toglucose that yeasts can further ferment into ethanol. This production ofethanol from readily available sources of cellulose can provide astable, renewable fuel source. The use of ethanol as a fuel has manyadvantages compared to petroleum fuel products such as a reduction inurban air pollution, smog, and ozone levels, thus enhancing theenvironment. Moreover, ethanol as a fuel source would reduce thereliance on foreign oil imports and petrochemical supplies.

But the major rate limiting step to ethanol production from biomass isthe insufficient amount of β-glucosidase in the system to efficientlyconvert cellobiose to glucose. Therefore, a cellulase composition thatcontains an enhanced amount of β-glucosidase would be useful in ethanolproduction. contrarily, in some cases, it is desirable to produce acellulase composition which is deficient in, and preferably free ofβ-glucosidase. Such compositions would be advantageous in the productionof cellobiose and other cellooligosaccharides.

β-glucosidases are present in a variety of prokaryotic organisms, aswell as eukaryotic organisms. The gene encoding β-glucosidase has beencloned from several prokaryotic organisms and the gene is able to directthe synthesis of detectable amounts of protein in E. coli withoutrequiring extensive genetic engineering, although, in some cases,coupling with a promotor provided by the vector is required. However,β-glucosidases are not produced by such organisms in commerciallyfeasible amounts.

Furthermore, such prokaryotic genes often cannot be expressed anddetected after transformation of the eukaryotic host. Thus, in order touse fungal strains, fungal genes would have to be cloned using methodsdescribed herein or by detection with the T. reesei bg11 gene by nucleicacid hybridization.

The contribution and biochemistry of the β-glucosidase component incellulose hydrolysis is complicated by the apparent multiplicity ofenzyme forms associated with T. reesei and other fungal sources (Enariet al, "Purification of Trichoderma reesei and Aspergillus nigerβ-glucosidase", J. Appl. Biochem., Vol. 3, pp. 157-163 (1981); Umile etal, "A constitutive, plasma membrane bound β-glucosidase in Trichodermareesei", FEMS Microbiology Letters, Vol. 34, pp. 291-295 (1986); Jacksonet al, "Purification and partial characterization of an extracellularβ-glucosidase of Trichoderma reesei using cathodic run, polyacrylamidegel electrophoresis", Biotechnol. Bioeng., Vol. 32, pp. 903-909 (1988)).These and many other authors report β-glucosidase enzymes ranging insize from 70-80 Kd and in pI from 7.5-8.5. More recent data suggeststhat the extracellular and cell wall associated forms of β-glucosidaseare the same enzyme (Hofer et al, "A monoclonal antibody against thealkaline extracellular β-glucosidase from Trichoderma reesei: reactivitywith other Trichoderma β-glucosidases", Biochim. Biophys. Acta, Vol.992, pp. 298-306 (1989); Messner and Kubicek, "Evidence for a single,specific β-glucosidase in cell walls from Trichoderma reesei QM9414",Enzyme Microb. Technol., Vol. 12, pp. 685-690 (1990)) and that thevariation in size and pI is a result of post translational modificationand heterogeneous methods of enzyme purification. It is unknown whetherthe intracellular β-glucosidase species with a pI of 4.4 and an apparentmolecular weight of 98,000 is a novel β-glucosidase (Inglin et al,"Partial purification and characterization of a new intracellularβ-glucosidase of Trichoderma reesei", Biochem. J., Vol. 185, pp. 515-519(1980)) or a proteolytic fragment of the alkaline extracellularβ-glucosidase associated with another protein (Hofer et al, supra).

Since a major part of the detectable β-glucosidase activity remainsbound to the cell wall (Kubicek, "Release of carboxymethylcellulase andβ-glucosidase from cell walls of Trichoderma reesei", Eur. J. Appl.Biotechnol., Vol. 13, pp. 226-231 (1981); Messner and Kubicek, supra;Messner et al, "Isolation of a β-glucosidase binding and activatingpolysaccharide from cell walls of Trichoderma reesei", Arch. Microbiol.,Vol. 154, pp. 150-155 (1990)), commercial preparations of cellulase arethought to be reduced in their ability to produce glucose because ofrelatively low concentrations of β-glucosidase in the purified cellulasepreparation.

To overcome the problem of β-glucosidase being rate limiting in theproduction of glucose from cellulose using cellulase produced by afilamentous fungi, the art discloses supplementation of the cellulolyticsystem of Trichoderma reesei with the β-glucosidase of Aspergillus andthe results indicate an increase in rate of saccharification ofcellulose to glucose. Duff, Biotechnol Letters, 7, 185 (1985). Culturingconditions of the fungi have also been altered to increase β-glucosidaseactivity in Trichoderma reesei as illustrated in Sternberg et al, Can.J. Microbiol., 23, 139 (1977) and Tangnu et al, Biotechnol. Bioeng., 23,1837 (1981), and mutant strains obtained by ultraviolet mutation havebeen reported to enhance the production of β-glucosidase in Trichodermareesei. Although these aforementioned methods increase the amount ofβ-glucosidase in Trichoderma reesei, the methods lack practicality and,in many instances, are not commercially feasible.

A genetically engineered strain of Trichoderma reesei or otherfilamentous fungi that produces an increased amount of β-glucosidasewould be ideal, not only to produce an efficient cellulase system, butto further use the increased levels of expression of the ball gene toproduce a cellulase product that has increased cellulolytic capacity.Such a strain can be feasibly produced using transformation.

But, in order to transform mutant strains of Trichoderma reesei or otherfilamentous fungi, the amino acid sequence of the ball gene ofTrichoderma reesei or the other filamentous fungi must be firstcharacterized so that the bg11 gene can be cloned and introduced intomutant strains of Trichoderma reesei or other filamentous fungi.

Additionally, once the bg11 gene has been identified, information withinlinear fragments of the ball gene can be used to prepare strains ofTrichoderma reesei and other filamentous fungi which produce cellulasecompositions free of β-glucosidase.

Accordingly, this invention is directed, in part, to thecharacterization of the bg11 gene that encodes for extracellular or cellwall bound β-glucosidase from Trichoderma reesei and other filamentousfungi. This invention is further directed to the cloning of the bg11gene into a plasmid vector that can be used in the transformationprocess, and to introduce the ball gene into the Trichoderma reesei orother filamentous fungi genome in multiple copies, thereby generatingtransformed strains which produce a cellulase composition having asignificant increase in β-glucosidase activity. Moreover, cellulasecompositions that contain increased cellulolytic capacity are alsodisclosed.

This invention is further directed, in part, to the deletion ordisruption of the ball gene from the Trichoderma reesei or otherfilamentous fungi genome. In addition, altered copies of the bg11 genewhich may change the properties of the enzyme can be reintroduced backinto the Trichoderma reesei or other filamentous fungi genome.

SUMMARY OF THE INVENTION

The amino acid sequence of the extracellular or cell wall boundβ-glucosidase protein from Trichoderma reesei has now been obtained insufficient detail to enable the bg11 gene to be cloned into a suitableplasmid vector. The plasmid vector can then be used to transform strainsof filamentous fungi to produce transformants which have multiple copiesof the bg11 gene introduced therein.

Accordingly, in one of its process aspects, the present inventionrelates to a process for expressing enhanced extracellular β-glucosidasein a filamentous fungus comprising expressing a fungal DNA sequenceencoding enhanced β-glucosidase in a recombinant host microorganism,said recombinant host microorganism being a filamentous fungustransformed with an expression vector containing said DNA sequence.

In another process aspect, the present invention relates to a processfor expressing cellulases from a β-glucosidic filamentous fungi whichare free of extracellular β-glucosidase.

In yet another process aspect, the present invention relates to aprocess for expressing an altered extracellular β-glucosidase in afilamentous fungus.

In another aspect, the present invention is directed to the amino acidsequence of extracellular β-glucosidase from Trichoderma reesei.

In yet another aspect, the present invention is directed to use of anucleic acid fragment comprising the entire or partial nucleotidesequence of the T. reesei extracellular β-glucosidase gene as a probe toidentify and clone out the equivalent bg11 gene from other β-glucosidicfilamentous fungi.

In one of its composition aspects, the present invention is directed tonovel and useful transformants of Trichoderma reesei, which can be usedto produce fungal cellulase compositions, especially fungal cellulasecompositions enriched in β-glucosidase or deleted of β-glucosidase. Alsocontemplated in the present invention is the alteration of the bg11 geneand the introduction of the altered bg11 gene into T. reesei to producetransformants which can also be used to produce altered fungal cellulasecompositions.

In another composition aspect, the present invention is directed tofungal cellulase compositions prepared via the transformed Trichodermareesei strains.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleotide sequence and deduced primary amino acidstructure of the entire T. reesei bg11 gene.

FIG. 2 is a schematic representation of the vector pSASβ-glu.

FIG. 3A is a figurative representation of the vector pSASΔβGlu bal pyr(Δ36).

FIG. 3B is a figurative representation of the vector DUCΔβ-Glu A/R pyr(Δ12).

FIG. 4 represents a Northern blot of total RNA isolated from thetransformed strains of Trichoderma reesei following induction withsophorose using the probes of cbh2 and a 700 bp fragment of bg11 cDNA.

FIG. 5A represents an autoradiograph of a Southern blot of T. reesei DNAillustrating the presence of β-glucosidase gene in wild type T. reesei(RL-P37) compared to strains of T. reesei genetically modified so as tonot include the β-glucosidase gene (Δ12 and Δ36).

FIG. 5B represents an autoradiograph of a Northern blot of T. reesei RNAillustrating the expression of β-glucosidase gene in wild type T. reesei(RL-P37) compared to strains of T. reesei genetically modified so as tonot include the β-glucosidase gene (Δ12 and Δ36).

FIG. 5C represents an analysis of the proteins expressed by P37 (wildtype), Δ12, and Δ36 strains of Trichorderma reesei and illustrates theabsence of β-glucosidase in the proteins expressed by Δ12 and Δ36strains Trichoderma reesei.

FIG. 6 represents an autoradiograph of Hind III digested genomic DNAfrom a T. reesei overproducing strain and transformants of pSASβ-Glu,blotted and probed with the 700 bp β-Glu probe.

FIG. 7 represents a curve illustrating Avicel hydrolysis using thedosage, substrate:enzyme of 80:1 from an enriched recombinantβ-glucosidase composition produced by the present invention.

FIG. 8 represents a curve illustrating PSC hydrolysis using the dosage,substrate:enzyme of 300:1 from an enriched recombinant β-glucosidasecomposition produced by the present invention.

FIG. 9 represents a curve illustrating the rate of hydrolysis of acellulosic diaper derived fibers using an enriched recombinantβ-glucosidase composition produced by the present invention.

FIGS. 10A and 10B are autoradiographs of Aspergillus nidulans,Neurospora crassa, Humicola grisea genomic DNA digested with Hind IIIand Eco RI, blotted and probed with a DNA fragment containing the bg11gene of Trichoderma reesei.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

As used herein, the term "enhanced extracellular β-glucosidase" or"enhanced β-glucosidase" means that at least one additional copy of agene encoding for extracellular β-glucosidase has been introduced intothe genome.

The term "devoid of the bg11 gene" means either that the bg11 gene hasbeen deleted from the genome and therefore cannot be expressed by therecombinant host microorganism; or that the bg11 gene has been disruptedin the genome so that a functional extracellular β-glucosidase enzymecannot be produced by the recombinant host microorganism.

The term "altered β-glucosidase" or "altered β-glucosidase gene" meansthat the amino acid sequence of the expressed protein has been alteredby removing, adding, and/or manipulating the nucleic acid sequence ofthe gene or the amino acid sequence of the protein.

The term "by recombinant means" denotes that a microorganism has beentransformed with a DNA molecule created in a test-tube by ligatingtogether pieces of DNA that are not normally contiguous.

The term "cellulase free of extracellular β-glucosidase" refers to acellulase composition which does not contain functional extracellularβ-glucosidase enzyme. Such compositions are preferably prepared byculturing a filamentous fungi wherein the β-glucosidase gene has beeneither deleted or disrupted. Preferably, these compositions are preparedby culturing a filamentous fungi wherein the β-glucosidase gene has beendeleted.

The term "filamentous fungi" means any and all art recognizedfilamentous fungi.

The term "β-glucosidic filamentous fungi" refers to those filamentousfungi which produce a cellulase composition containing β-glucosidase.

The term "cellooligosaccharide" refers to those oligosaccharide groupscontaining from 2-8 glucose units having β-1,4 linkages. Suchcellooligosaccharides include cellobiose (diglucose having a β-1,4-linkage) and are preferably derived from cellulose.

More specifically, the present invention relates to the isolation andcharacterization of the bg11 gene coding for the extracellular or cellwall bound protein from Trichoderma reesei (sometimes referred to as "T.reesei") and the specific nucleotide and amino acid sequence of thisgene. The bg11 gene is cloned into plasmid vectors, which are furtherused to produce transformed strains of T. reesei and other filamentousfungi having extra copies of the bg11 gene inserted therein. Thesetransformants are then used to produce cellulase compositions havingincreased β-glucosidase activity and thus enhanced cellulolyticdegradation.

Besides enhancing cellulolytic degradation by inserting extra copies ofthe bg11 gene into T. reesei strains, it is also contemplated by thepresent invention to produce transformed strains that are completelydevoid of the bg11 gene.

Also contemplated by the present invention is the manipulation of theamino acid sequence in the bg11 gene itself. Alteration of the activesites on this enzyme may lead to a variety of different changes incatalytic conversion. For example, since β-glucosidase has bothhydrolase and transferase activity, alteration of the amino acidsequence may result in the removal of hydrolase activity and an increasein transferase activity and, thus, facilitate the synthesis of B 1-4oligo-dextrins. Moreover, manipulation of the amino acid sequence ofβ-glucosidase may result in further changes in the system, such asdifferent pH optima, different temperature optima, altered catalyticturn over rate (Vmax), altered affinity (Km) for cellobiose leading toan increased affinity for cellobiose or a decreased affinity forcellobiose resulting in a slower or zero rate of reaction, alteredproduct inhibition profile such that lower or higher levels of glucosewill inhibit β-glucosidase activity, and the like.

Moreover, a nucleic acid fragment containing the entire nucleotidesequence of the extracellular β-glucosidase gene in T. reesei or aportion thereof can also be labeled and used as a probe to identify andclone out the equivalent bg11 gene in other filamentous fungi.

Generally, the present invention involves the isolation of the bg11 genefrom T. reesei by identifying a 700 bp cDNA fragment of the gene whichis then used as a probe to identify a single T. reesei fragmentcontaining the bg11 gene which was subsequently cloned. Because of thespecies homology of the bg11 gene, a probe employing a fragment of thebg11 gene of T. reesei can be employed to identify the bg11 gene inother cellulolytic microorganisms and, it is understood that thefollowing description for T. reesei could also be applied to otherβ-glusosidic filamentous fungi.

In the case of T. reesei, this 6.0 kb fragment is then cloned into a pUCplasmid and a series of mapping experiments are performed to confirmthat the entire bg11 gene is contained in this fragment. The nucleotidesequence is then determined on both strands and the position of twointrons can be confirmed by sequence analysis of bg11 cDNA subclonesspanning the intron/exon boundaries. After isolation of the bg11 gene,additional bg11 gene copies are then introduced into T. reesei or otherfilamentous fungal strains to increase the expression of β-glucosidase.

In contrast, the entire bg11 gene can also be deleted from the genome ofT. reesei and other β-glucosidic filamentous fungi, thereby producingtransformants that express cellulases free of β-glucosidase.

The isolation of the bg11 gene from T. reesei involves the purificationof extracellular β-glucosidase, chemical and proteolytic degradation ofthis protein, isolation and determination of the sequence of theproteolytic fragments and design of synthetic oligomer DNA probes usingthe protein sequence. The oligomeric probes are then further used toidentify a 700 bp β-glucosidase cDNA fragment which can be labeled andemployed to later identify a fragment that contains the entire bg11 genewithin the fragment from digested genomic DNA from T. reesei.

To identify a feasible cDNA fragment that can be used as a probe forfuture analysis, total RNA is first isolated from T. reesei mycelia andpolyadenylated RNA isolated therefrom. The polyadenylated RNA is thenused to produce a cDNA pool which is then amplified using specificoligonucleotide primers that amplify only the specific cDNA fragmentencoding the T. reesei bg11 gene.

More specifically, total RNA is first isolated from a starting strain ofT. reesei. The starting strain employed in the present invention can beany T. reesei cellulase overproduction strain that is known in the art.This cellulase producing strain is generally developed by ordinarymutagenesis and selection methods known in the art from any T. reeseistrain. Confirmation that the selected strain overproduces cellulasescan be performed by using known analysis methods. A preferred strain isRLP37 which is readily accessible.

A mycelial inoculum from the T. reesei over production strain, grown inan appropriate growth medium, is added to a basal medium and incubatedfor a period of between 50-65 hours at a temperature between 25° C. to32° C., preferably 30° C. Fresh basal medium can be replaced during thisincubation period. The culture medium is then centrifuged, and themycelia is isolated therefrom and washed. The mycelia is thenresuspended in a buffer to permit growth thereof and 1 mM sophorose (aβ,1-2 dimer of glucose) is added to the mycelia to induce the productionof cellulase enzymes. The mycelia preparation is then incubated for anadditional time period, preferably 18 hours at 30° C. prior toharvesting.

Total RNA can be isolated from the mycelia preparation by a variety ofmethods known in the art, such as proteinase K lysis, followed byphenol:chloroform extraction, guanidinium isothiocyanate extraction,followed by cesium chloride gradients, guanidine hydrochloride andorganic solvent extraction, and the like. It is preferable to isolatetotal RNA via the procedure described by Timberlake et al in"Organization of a Gene Cluster Expressed Specifically in the AsexualSpores of A. nidulans," Cell, 26, pp. 29-37 (1981). The mycelia isisolated from the culture medium via filtration. Then the RNA isextracted from the mycelia by the addition of an extraction buffer,TE-saturated phenol and chloroform. The aqueous phase is removed and theorganic phase is reextracted with the extraction buffer alone by heatingthe extraction mixture in a water bath at a temperature between about60° C. to 80° C., preferably 68° C. to release the RNA trapped inpolysomes and at the interface. All of the extracted aqueous phases arethen pooled, centrifuged and reextracted with phenol-chloroform untilthere is no longer any protein at the interface. The RNA is furtherprecipitated with 0.1 volume of 3 M sodium acetate and 2 volumes of 95%ethanol and pelleted via centrifugation before it is resuspended inDEP-water containing an RNase inhibitor.

The total RNA is then fractionated on 1% formaldehyde-agarose gels,blotted to Nytran™ membranes, and probed using a fragment of the T.reesei cbh2 gene to determine whether the genes encoding the enzymes ofthe cellulase system in the T. reesei preparation are indeed induced byaddition of the sophorose. Basically, the probe used in the presentinvention is derived from a CBH II clone produced by methods known inthe art. For more specific detail of how the clone was produced see Chenet al, "Nucleotide Sequence and Deduced Primary Structure ofCellobio-hydrolase II from Trichoderma reesei," Bio/Technology, Vol. 5(March 1987). Site directed mutagenesis was performed on the CBH IIclone and a bgl II site was placed at the exact 5' end of the openreading frame and a Nhe I site at the exact 3' end. The Bgl II and Nhe Irestriction fragment containing CBH II coding sequence was furthercloned into a pUC218 phagemid. The CBH II gene was further cut and gelisolated prior to adding a label.

The results of the Northern blot of T. reesei RNA probed with the cbh2probe indicated that the level of cbh2 specific mRNA reached a peak at14-18 hours post induction. From this data it can be inferred that theentire cellulase complex including β-glucosidase is induced at thistime. The total RNA from 14, 18 and 22 hours is then pooled.

After pooling the specific fractions of total RNA, polyadenylated mRNAis further isolated from the total RNA. Postranscriptionalpolyadenylation is a common feature of the biogenesis of most eukaryoticmRNAs. The newly synthesized mRNAs have long poly(A) tracts which tendto shorten as mRNAs age. The newly synthesized polyadenylated mRNA isfurther isolated from total RNA by methods known in the art. Thesemethods include the use of oligo(dT)-cellulose, poly(U) Sepharose,adsorption to and elution from poly(U) filters or nitrocellulosemembrane filters, and the like. It is preferable to use oligo(dT)cellulose chromatography in isolating MRNA following the proceduredescribed by Sambrook et al, Molecular Cloning, A Laboratory Manual, 2ndEdition, Cold Spring Harbor Laboratory Press (1989). More specifically,fractions of total RNA are run through the chromatographic resin, andmRNA is eluted therefrom with an elution buffer. The RNA which binds tothe column is enriched for RNAs containing poly(A) tails and, therefore,eliminates contaminants, such as rRNA and partially degraded mRNAs. Itis important that the purification be carried out successfully such thatwhen cDNA is synthesized from the mRNA, higher yields of mRNA copies andless spurious copying of non-messenger RNAs occurs.

Total RNA and polyadenylated RNA from the preparations were furtherfractionated on 1% formaldehyde gels, blotted to Nytran^(R) membranesand analyzed to confirm that the enzymes in the cellulase complex werebeing induced as polyadenylated mRNA.

After isolating polyadenylated mRNA from total RNA, complementary DNA orcDNA is synthesized therefrom. The first strand of cDNA is synthesizedusing the enzyme RNA-dependent DNA polymerase (reverse transcriptase) tocatalyze the reaction. Avian reverse transcriptase which is purifiedfrom the particles of an avian retrovirus or murine reversetranscriptase, which is isolated from a strain of E. coli that expressesa cloned copy of the reverse transcriptase gene of the Moloney murineleukemia virus can be used in the present invention. However, it ispreferable to use the Moloney murine leukemia virus (M-MLV) reversetranscriptase to synthesize first strand cDNA from the polyadenylatedmRNA population. The amount of cloned M-MLV reverse transcriptaserequired may vary depending on the amount of polyadenylated mRNA used inthe synthesis reaction. Usually, about 200 U/μl of the reversetranscriptase is used per 2 to 10 μg of mRNA per reaction.

Also present in the synthesis mixture is a primer to initiate synthesisof DNA. For cloning of cDNAs, any primer can be used, but it ispreferable to use oligo(dT) containing 12-18 nucleotides in length,which binds to the poly(A) tract at the 3' terminus of eukaryoticcellular mRNA molecules. The primer is added to the reaction mixture inlarge molar excess so that each molecule of mRNA binds several moleculesof oligo(dT)₁₂₋₁₈. It is preferable to use about 12.5 μg of primerhaving a concentration of 0.5 mg/ml.

Besides the enzyme and primer, a buffer and dNTP mix containing DATP,dCTP, dGTP, and dTTP at a final concentration of 500 μM each usuallycompletes the reaction cocktail. Any buffer can be used in the presentinvention for first strand cDNA synthesis that is compatible with thissynthesis. It is preferable to use a buffering system consisting of 250mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl₂, and 50 mM dithiothreitol.Generally, about 500 μl of buffer completes the synthesis solution.

After the first strand is synthesized, the second strand of cDNA may besynthesized by a variety of methods known in the art, such ashairpin-primed synthesis by denaturing the cDNA:mRNA complex, adding theKlenow fragment of E.coli DNA polymerase or reverse transcriptase, andthen digesting the hairpin loop with nuclease S1 to obtain adouble-stranded cDNA molecule, the Okayama and Berg method, the Gublerand Hoffman method, and the like. The Okayama and Berg method uses E.coli RNase H to randomly nick the mRNA, and the RNA is replaced in thenick translation reaction by catalysis with E. coli DNA polymerase I. Inthe Okayama and Berg method, mRNA is used to prime DNA synthesis by theE. coli DNA polymerase I.

The preferred method to synthesize the second strand of cDNA is amodified method of the Gubler and Hoffman procedure. This procedure usesE. coli RNase H, DNA Polymerase I, and DNA Ligase to form the secondstrand. Actually, two different methods of proceeding with the secondstrand synthesis can be used in the present invention. The firstprocedure uses RNase H to attack the RNA:DNA hybrid in a random fashion,producing nicks in addition to those produced by reverse transcriptase.If too many nicks are introduced into the RNA at the 51 end of themessage before second strand synthesis commences, fragments may beproduced that are too short to remain hybridized; thus, they will not beable to serve as primers. In addition, the 5'-most RNA oligomer whichprimes second strand DNA synthesis will continue to be degraded untilonly two ribonucleotides remain at the 5' end of the second strand DNA.These are substrates for the polymerase I RNase H activity, and theremaining nucleotides will be removed. This leaves the 3' end of thefirst strand cDNA single stranded, making it a substrate for the 3'exonuclease activity of Polymerase I. The result is a population ofcDNAs, which are blunt-ended.

An alternative method relies on M-MLV reverse transcriptase to producenicks 10 to 20 bases from the 5' end of the RNA in the hybrid. DNApolymerase I is then used for synthesis. Generally, about 500 units at aconcentration of 10 U/μl of DNA polymerase I is used. After secondstrand synthesis, RNase H is added after removal of the DNA polymerase Ito produce a duplex, which is entirely DNA, except for the survivingcapped RNA 5' oligonucleotide.

The second-strand synthesis by either procedure set forth above usuallytakes place in the presence of a buffer and dNTP mix. Any bufferingsystem that is known in the art for second strand cDNA synthesis can beused; however, it is preferable to use a buffering system containing 188mM Tris-HCl, pH 8.3, 906 mM KCl, 100 mM (NH₄)₂ SO₄, 46 mM MgCl₂, 37.5 mMdithiothreitol, and 1.5 mM HAD. The dNTP mix preferably contains 10 mMDATP, 10 mM dCTP, 10 mM dGTP, and 10 mM dTTP.

The second strand synthesis is carried out under known procedures setforth in the art. The preferred methods and reagents used to synthesizecDNA in the present invention are the BRL cDNA Synthesis System^(R)(Bethesda Research Laboratories, Gaithersburg, Md.) and the LibrariumSystem (Invitrogen, San Diego, Calif.).

At this point a pool of cDNAs, a small portion of which code for thebg11 gene, is present after second strand synthesis. Since amplificationof only the specific bg11 gene fragment in the cDNA pool is crucial forthe isolation of the β-glucosidase gene, specific primers were designedto amplify the cDNA fragment encoding the T. reesei bg11 gene in thepolymerase chain reaction (PCR). The primers used are degenerate primersdesigned to hybridize to the cDNA of the bg11 gene encoding theN-terminus and an internal CNBr fragment.

In general, it is difficult to isolate the bg11 gene because the aminoacid sequence of the protein does not contain sufficient amino acidswhich are coded for by unique nucleic acid triplets and thus anyoligonucleotide used would be too degenerate to specifically amplify thebg11 gene in the PCR reaction. However, in this invention, primers weredesigned by examining the amino acids of the region targeted foramplification of mature β-glucosidase and choosing regions, which willrequire a reduced degree of degeneracy in the genetic code. Codon biasin T. reesei for various other cellulase genes such as cbh1, cbh2, eg11,and the like was also taken into account when designing theoligonucleotide primers. More specifically, codon bias is based onvarious genes in the strain T. reesei which display a preferrednucleotide triplet encoding different amino acids. By analyzing thiscodon bias one can determine that a particular nucleotide sequencecoding for an amino acid would be preferred. For example, the cbh1, cbh2and eg1 genes from T. reesei prefer the CCU coding for the amino acidproline. Thus, when designing an oligonucleotide probe, the CUG sequencewould be the preferred choice for leucine, rather than the othertriplets (CUU, CUC, CUA, UUA and CUG) which code for leucine.

Furthermore, after selection of an N-terminal region and an internalregion as primers for amplification purposes, the primers were designedby inserting a non-specific base inosine into the wobble position of theprimer for the N-terminus and using a pool of sixteen variable primersequences for the internal primer. Basically, the creation of degenerateprimers is described by Compton in "Degenerate Primers For DNAAmplification" and Lee et al in "cDNA Cloning Using Degenerate Primers"in PCR Protocols: A Guide to Methods and Applications, published byAcademic Press (1990).

Using the primers described above, the cDNA sequences encoding the aminoterminal region of the bg11 gene is then selectively amplified usingPCR. The amplification method consists of an initial, denaturing cycleof between about 5 to 15 minutes at 95° C., followed by a 1-7 minutesannealing step at a temperature between 35° C. and 55° C. and preferablybetween 45° C. and 55° C. and a 5-15 minutes polymerization cycle at 65°C. It is preferable, however, to use a 10 minute initial denaturingcycle, followed by 2 minutes of annealing at 50° C. and a 10 minute, andpreferably a 30 minute polymerization cycle at the aforedescribedtemperatures.

The amplified fragment is then identified via gel electrophoresis as a700 bp cDNA segment. The amplified pool of cDNAs is then furtherfractionated on a polyacrylamide gel to obtain a more purified 700 bpcDNA fragment for cloning purposes. After elution of the 700 bpfragments from the gel, the 700 bp cDNA fragments are then cloned intophagemid vectors. Any cloning vector can be used to clone the cDNA bg11gene fragments, such as pUC18, pUC19, pUC118, pUC119, pBR322, pEMBL,pRSA101, pBluescript, and the like. However, it is preferable to use thecloning vectors pUC218 and pUC219, which are derived from pUC18 andpUC19 by insertion of the intergenic region of M13. The cloning vectorswith the cDNA fragments containing the bg11 gene are then used totransform E. coli strain JM101. After transformation, positive coloniescontaining the bg11 gene were identified and DNA isolated therefromusing chloroform:phenol extraction and ethanol precipitation methods.

The nucleotide sequence of the subcloned cDNA 700 bp fragment is thendetermined by the dideoxy chain termination method described by Sangeret al using a Sequenase^(R) reagent kit provided by U.S. Biochemicals.

From this nucleotide sequence it was determined that the subcloned 700bp cDNA segment contained an open reading frame encoding 150 amino acidsthat overlapped a number of other sequenced peptides that were obtainedfollowing CNBr and proteolytic degradation of purified T. reeseiβ-glucosidase. Thus, it was confirmed that the cloned sequences encodedfor the extracellular T. reesei β-glucosidase protein.

The cloning of the genomic version of the entire β-glucosidase gene wasthen undertaken by labelling the 700 bp bg11 cDNA fragment with ³² pusing the methods to label oligonucleotides described by Sambrook et al,supra. This probe is used to identify a 6.0 kb band on a Southern blotof Hind III digested genomic DNA from T. reesei.

The genomic DNA from T. reesei is prepared for Southern blot analysis bydeproteinizing the genomic DNA, followed by treatment with ribonucleaseA. The prepared genomic DNA is then cut with one of a variety ofrestriction enzymes such as Eco RI, Hind III and the like, run on a gel,Southern blotted and hybridized with the 700 bp cDNA labelled fragmentof the bg11 gene. From this analysis, it was determined that Hind IIIwas the restriction enzyme of choice that can be used to clone the bg11gene.

Hind III is then added to genomic DNA from the strain T. reesei and DNAis extracted therefrom. A sample from this digestion is run on anagarose gel and fractionated electrophoretically. The gel is thenSouthern blotted and probed with the 700 bp cDNA probe. A 6.0 kb bandwas then identified on the Southern blot of Hind III digested genomicDNA. The remaining Hind III digested genomic DNA was then subjected topreparative gel electrophoresis and DNA ranging in size from about 5.0kb to 7.0 kb was eluted therefrom and cloned into a phagemid vector andused to transform E. coli JM101 to create a library. Any phagemid vectorcan be used such as those described above, however it is preferable touse pUC218. The colonies that resulted from the transformation were thensubjected to colony hybridization using the 700 bp cDNA fragment as aprobe to identify those colonies that contained the cloned genomic DNAcoding for bg11 . The positive colonies from the transformation are thenpicked and the DNA isolated therefrom by methods known in the art.

The isolated DNA from such a positive colony is then digested withvarious restriction enzymes, both singly and in various combinations,and subjected to agarose gel electrophoresis. The resultant bandingpattern is then used to construct a restriction map of the cloned 6.0 kbgenomic DNA from T. reesei. Enzymes used in the digestion include EcoRI, Sst I, Kpn I, Sma I, Bam HI, Xho 1, Bgl II, Cla I, Xba I, Sal I, PstI, Sph I, Hind III, Bal I, Pvu II and the like.

The same gel is then subject to Southern blot analysis using the same700 bp bg11 cDNA as a probe to identify which genomic restrictionfragments shared homology with the bg11 cDNA. Since the position ofthese homologous fragments can be determined relative to the restrictionmap of the 6.0 kb genomic fragment and also since the size of theβ-glucosidase protein (74 kd) gives an estimated length of the gene as2.1 kb (because average molecular weight of an amino acid is 105daltons, a 74 kd protein contains on average 705 amino acids, which inturn is equal to 2,115 bp), then the mapping experiments confirmed thatthe entire bg11 gene is contained on the genomic Hind III clone.

Pvu II and Bal I restriction fragments ranging in size from 600 bp to1500 bp hybridized with the 700 bp cDNA bg11 clone and were thus chosenfor subcloning into pUC218 phagemids. The nucleotide sequence wasdetermined using the methods of Sanger et al, described above. The PvuII and Bal I subclones were sequenced and the overlapping sequences ofthe subclones aligned until a single contiguous sequence totaling 3033bp was obtained within which the nucleotide sequence of the bg11 genewas determined on both strands and the position of two small introns wasinferred by homology to introns of other genes of filamentous fungi. Theamino acid sequence is also deduced as set forth in FIG. 1.

The nucleotide sequence and deduced primary amino acid sequence of theentire T. reesei bg11 gene is set forth in FIG. 1 (SEQ ID NO:1). Thepredicted molecular weight of the encoded β-glucosidase protein is74,341. A 31 amino acid peptide precedes the mature amino terminus ofβ-glucosidase as deduced from the amino terminal peptide sequence.Within this peptide, there are three potential signal peptidaserecognition sites consisting of Ala-X-Ala.

The primary amino acid sequence of β-glucosidase (SEQ ID NO:2) shows 7potential N-linked glycosylation sites at positions 208, 310, 417, and566, which shows the consensus Asn-X-Ser/Thr-X where X is not a proline.However, sites at positions 45, 566, and 658 have a proline residue inthe consensus sequence and may or may not be glycosylated.

No unusual codon bias is observed in the bg11 gene when compared toother cellulase genes. The bg11 coding region is interrupted by twoshort introns of 70 bp and 64 bp, respectively. Both introns have splicesite donor, splice acceptor, and lariat branch acceptor sites that showhomology to the consensus splice signals emerging from T. reesei andother filamentous fungi.

Since the bg11 gene from the T. reesei strain is identified and can becloned, the next step is to produce a transformant that has extra copiesof the bg11 gene.

A selectable marker must first be chosen so as to enable detection ofthe transformed filamentous fungus. Different selectable markers may beused including argB from A. nidulans or T. reesei, amdS from A.nidulans, pyr4 from Neurospora crassa, A. nidulans or T. reesei, andpyrG from Aspergillus niger. The selectable marker can be derived from agene, which specifies a novel phenotype, such as the ability to utilizea metabolite that is usually not metabolized by the filamentous fungi tobe transformed or the ability to resist toxic shock effects of achemical or an antibiotic. Also contemplated within the presentinvention are synthetic gene markers that can be synthesized by methodsknown in the art. Transformants can then be selected on the basis of theselectable marker introduced therein. Because T. reesei does not containthe amds gene, it is preferable to use the amds gene in T. reesei as aselectable marker that encodes the enzyme acetamidase, which allowstransformant cells to grow on acetamide as a nitrogen source. In thecase where the bg11 gene is deleted from T. reesei, it is preferable touse the pyrG gene as a selectable marker.

The host strain used should be mutants of the filamentous fungi whichlack or have a nonfunctional gene or genes corresponding to theselectable marker chosen. For example, if the selectable marker of argBis used, then a specific arg mutant strain is used as a recipient in thetransformation procedure. Other examples of selectable markers that canbe used in the present invention include the genes trp, pyr4, pyrG,trp1, oliC31, Bm1, pkiA, niaD, leu, and the like. The correspondingrecipient strain must, therefore, be a mutant strain such as trp⁻ ' pyr⁻' leu⁻ ' and the like.

The mutant strain is derived from a starting host strain, which is anyfilamentous fungi strain. However, it is preferable to use a filamentousfungi over-producing mutant strain and particularly, a T. reeseioverproducing strain described previously, since this strain secreteshigh amounts of proteins and, in particular, high amounts of cellulaseenzymes. The selected mutant strain is then used in the transformationprocess. The preferred strain of T. reesei for use in deleting the bglIgene is RLP37 pyrG69, a uridine auxotroph.

The mutant strain of the selected filamentous fungi can be prepared by anumber of techniques known in the art, such as the filtration enrichmenttechnique described by Nevalainen in "Genetic improvement of enzymeproduction in industrially important fungal strains", Technical ResearchCenter of Finland, Publications 26 (1985). Another technique to obtainthe mutant strain is to identify the mutants under different growthmedium conditions. For instance, the arg mutants can be identified byusing a series of minimal plates supplied by different intermediates inarginine biosynthesis. Another example is the production of pyr⁻ mutantstrains by subjecting the strains to fluoroorotic acid (FOA). Strainswith an intact pvr4 gene grow in an uridine medium and are sensitive tofluoroorotic acid, and, therefore, it is possible to select pyr4- mutantstrains by selecting for FOA resistance.

The chosen selectable marker is then cloned into a suitable plasmid. Anyplasmid can be used in the present invention for the cloning of theselectable marker such as pUC18, pBR322, and the like. However, it ispreferable to use pUC100. The vector is created by digesting pUC100 withthe restriction enzyme SmaI, and the 5' phosphate groups are thenremoved by digestion with calf alkaline phosphatase. The fragment vectoris then purified by gel electrophoresis followed by electroelution fromthe isolated gel slice. The amds gene from A. nidulans is isolated as a2.4 kb SstI restriction fragment following separation of the vectorsequences via known procedures such as those described by Hynes et al,Mol, Cell. Biol., 3, pp. 1430-1439 (1983). The 2.4 Kb SstI amdS fragmentand the 2.7 Kb pUC100 vector fragment are then ligated together, and theligation mix is then introduced into the E. coli host strain JM101.

Any plasmid can be used in the present invention for the insertion ofthe bg11 gene, but it is preferable to use the pSAS plasmid. pSASβ-gluis constructed by digesting pSAS with the restriction enzyme Hind IIIand purifying the linear fragment via gel electrophoreses andelectroelution. Into this Hind III treated pSAS vector fragment isligated the 6.0 Kb Hind III fragment of T. reesei genomic DNA thatcontained all of the coding region of the bg11 gene along with thesequences necessary for transcription and translation. FIG. 2illustrates the construction of pSASβ-glu.

It is also possible to construct vectors that contain at least oneadditional copy of the bg11 gene and to construct vectors in which theamino acid sequence of bg11 gene has been altered by known techniques inthe art such as site directed mutagenesis, PCR methods, and chemicalmutation methods.

In another embodiment, the bg11 gene of a β-glucosidic filamentous fungican be totally deleted and may be replaced with other known genes.Preferably, the replacing gene is homologous to the filamentous fungi sothat the resulting recombinant microorganism does not express anyheterologous protein. For example, potentially any T. reesei gene whichclones for a selected marker and which has been cloned and thusidentified in the genome, can replace the bg11 gene in T. reesei usingthe techniques described herein.

On the other hand, the replacing gene does not necessarily have to behomologous. Specifically, for the deletion of the bg11 gene in T.reesei, vectors containing heterologous gene which have been used areillustrated in FIGS. 3A and 3B. In FIG. 3B, a pUC218 vector plasmidhaving the Aspergillus niger pyrG gene inserted therein is illustrated.A 6.0 Kb genomic Hin dIII fragment, known to contain the entire bg11gene, is cloned into the polylinker of pUC218. The coding region for thebg11 gene is then removed from this plasmid using either unique Ballrestriction sites (FIG. 3A) or unique ApaI and EcoRV restriction sites(FIG. 3B) situated at the very 5' and 3' end of the bglI open readingframe and replaced with an isolated 2412 bp Hin dIII/Bam HI restrictionfragment containing the pyrG gene from Asperaillus niger. Allrestriction ends are made blunt by treatment with T4 DNA polymeraseprior to ligation using T4 DNA ligase.

After a suitable vector is constructed, it is used to transform strainsof filamentous fungi. Since the permeability of the cell wall infilamentous fungi (e.g., T. reesei) is very low, uptake of the desiredDNA sequence, gene or gene fragment is at best minimal. To overcome thisproblem, the permeability of the cell wall can be increased or the DNAcan be directly shot into the cells via a particle gun approach. In theparticle gun approach, the DNA to be incorporated into the cells iscoated onto micron size beads and these beads are literally shot intothe cells leaving the DNA therein and leaving a hole in the cellmembrane. The cell then self-repairs the cell membrane leaving the DNAincorporated therein. Besides this aforedescribed method, there are anumber of methods to increase the permeability of filamentous fungicells walls in the mutant strain (i.e., lacking a functional genecorresponding to the used selectable marker) prior to the transformationprocess.

One method involves the addition of alkali or alkaline ions at highconcentrations to filamentous fungi cells. Any alkali metal or alkalineearth metal ion can be used in the present invention; however, it ispreferable to use either CaCl₂ or lithium acetate and more preferable touse lithium acetate. The concentration of the alkali or alkaline ionsmay vary depending on the ion used, and usually between 0.05 M to 0.4 Mconcentrations are used. It is preferable to use about a 0.1 Mconcentration.

Another method that can be used to induce cell wall permeability toenhance DNA uptake in filamentous fungi is to resuspend the cells in agrowth medium supplemented with sorbitol and carrier calf thymus DNA.Glass beads are then added to the supplemented medium, and the mixtureis vortexed at high speed for about 30 seconds. This treatment disruptsthe cell walls, but may kill many of the cells.

Yet another method to prepare filamentous fungi for transformationinvolves the preparation of protoplasts. Fungal mycelium is a source ofprotoplasts, so that the mycelium can be isolated from the cells. Theprotoplast preparations are then protected by the presence of an osmoticstabilizer in the suspending medium. These stabilizers include sorbitol,mannitol, sodium chloride, magnesium sulfate, and the like. Usually, theconcentration of these stabilizers varies between 0.8 M to 1.2 M. It ispreferable to use about a 1.2 M solution of sorbitol in the suspensionmedium.

Uptake of the DNA into the host mutant filamentous fungi strain isdependent upon the concentration of calcium ion. Generally, betweenabout 10 mM CaCl₂ and 50 mM CaCl₂ is used in an uptake solution. Besidesthe need for calcium ions in the uptake solution, other items generallyincluded are a buffering system such as TE buffer (10 mM Tris, pH 7.4; 1mM EDTA) or 10 mM MOPS, pH 6.0 buffer (morpholinepropane-sulfonic acid),and polyethylene glycol (PEG). The polyethylene glycol acts to fuse thecell membranes, thus permitting the contents of the mycelium to bedelivered into the cytoplasm of the filamentous fungi mutant strain andthe plasmid DNA is transferred to the nucleus. This fusion frequentlyleaves multiple copies of the plasmid DNA tandemly integrated into thehost chromosome. Generally, a high concentration of PEG is used in theuptake solution. Up to 10 volumes of 25% PEG 4000 can be used in theuptake solution. However, it is preferable to add about 4 volumes in theuptake solution. Additives such as dimethyl sulfoxide, heparinspermidine, potassium chloride, and the like may also be added to theuptake solution and aid in transformation.

Usually a suspension containing the filamentous fungi mutant cells thathave been subjected to a permeability treatment or protoplasts at adensity of 10⁸ to 10⁹ /ml, preferably 2×10⁸ /ml, are used intransformation. These protoplasts or cells are added to the uptakesolution, along with the desired transformant vector containing aselectable marker and other genes of interest to form a transformationmixture.

The mixture is then incubated at 4° C. for a period between 10 to 30minutes. Additional PEG is then added to the uptake solution to furtherenhance the uptake of the desired gene or DNA sequence. The PEG may beadded in volumes of up to 10 times the volume of the transformationmixture, preferably, about 9 times. After the PEG is added, thetransformation mixture is then incubated at room temperature before theaddition of a sorbitol and CaCl₂ solution. The protoplast suspension isthen further added to molten aliquots of a growth medium. This growthmedium contains no uridine and selectively permits the growth oftransformants only. The subsequent colonies were transferred andpurified on a growth medium depleted of sorbitol.

At this stage, stable transformants can be distinguished from unstabletransformants by their faster growth rate and the formation of circularcolonies with a smooth rather than ragged outline on solid culturemedium. Additionally, in some cases, a further test of stability can bemade by growing the transformants on solid non-selective medium,harvesting the spores from this culture medium and determining thepercentage of these spores which will subsequently germinate and grow onselective medium.

In order to ensure that the transformation took place by theabove-described methods, further analysis is performed on thetransformants such as Southern blotting and autoradiography. Using thesame basic procedures set forth above, the entire bg11 gene can bedeleted from a vector and transformed into filamentous fungi strains orthe bg11 gene can be altered and transformed into filamentous fungistrains.

After confirmation that the transformed strains contained at least oneadditional copy of the bg11 gene, an altered bg11 gene or thetransformants contained a deleted bg11 gene, the strains are furthercultured under conditions permitting these transformants to propagate.The transformants can then be isolated from the culture media and usedin a variety of applications which are described below. Alternatively,the transformants can be further fermented and a recombinant fungalcellulase composition can be isolated from the culture media. Since, forexample, the transformants produced by the present invention can expressenhanced, deleted or altered extracellular β-glucosidase in thefermentation medium, fungal cellulase compositions can be isolated fromthe medium. Usually, the isolation procedure involves centrifuging theculture or fermentation medium containing the transformants andfiltering by ultrafiltration the supernatant to obtain a recombinantlyproduced fungal cellulase composition. Optionally, an antimicrobialagent can be further added to the composition prior to use in thevariety of applications described below. Examples of microbial agentsthat can be added are sodium azide, sodium benzoate and the like.

Confirmation that the transformants produced by the process of thepresent invention had enhanced activity on cellobiose, the followingexperiment was performed. In this experiment 50 mg of cellobiose whichwas suspended in 1.0 ml of phosphate buffer (pH 5.0) and was reactedwith the fermentation product produced by the transformant (65.5 mg/mlprotein) using a fermentation product from a normal nonmutant T. reeseistrain as a control (135.0 mg/ml protein). The results of cellobiaseactivity under conditions of initial rate, are set forth in Table Ibelow:

                  TABLE I                                                         ______________________________________                                                               Activity on Cellobiose                                                        μmole glucose                                       Product    Protein (mg/ml)                                                                           mg protein                                             ______________________________________                                        Control    135.0       6                                                      Product    65.5        33                                                     produced                                                                      by the                                                                        present                                                                       invention                                                                     ______________________________________                                    

The results from this experiment indicate that the fermentation productproduced by the transformants of the present invention has over fivetimes the specific activity on the substrate, cellobiose, compared to anonmutant T. reesei control strain.

Moreover, FIGS. 7 and 8 confirm that hydrolysis is enhanced for thesubstrates Avicel and PSC (note: PSC is a phosphoric acid swollencellulose obtained by treating Avicel with phosphoric acid) using 1.0%enzyme/substrate. In the experiment, PSC or Avicel was suspended in 2mls of 50 mM sodium acetate buffer, pH 4.8, and incubated at 40° undernon-agitated conditions for up to 24 hours. Soluble reducing sugar wasmeasured by the method of Nelson and Somogyi. From these figures it isfurther demonstrated that the enhanced recombinant β-glucosidasefermentation product produced from transformants according to thepresent invention, has an increased rate and extent of hydrolyticactivity on the various substrates compared to the standard Cyt-123control (on average 20% higher activity). The Cyt-123 control is theproduct obtained from a T. reesei cellulase over-production strainsubjected to fermentation on an industrial scale.

The enriched transformants can be used in a variety of differentapplications. For instance, some β-glucosidases can be further isolatedfrom the culture medium containing the enhanced transformants and addedto grapes during wine making to enhance the potential aroma of thefinished wine product. Yet another application can be to useβ-glucosidase in fruit to enhance the aroma thereof. Alternatively, theisolated recombinant fermentation product containing enhancedβ-glucosidase can be used directly in food additives or wine processingto enhance the flavor and aroma.

Since the rate of hydrolysis of cellulosic products is increased byusing the transformants having at least one additional copy of the bg11gene inserted into the genome, products that contain cellulose orheteroglycans can be degraded at a faster rate and to a greater extent.Products made from cellulose such as paper, cotton, cellulosic diapersand the like can be degraded more efficiently in a landfill. FIG. 9illustrates the use of an increased β-glucosidase preparation isolatedfrom the fermentation medium containing transformants having at leastone additional copy of the bg11 gene inserted into the genome comparedto a non-enhanced Cyt 123 standard (defined above) on a cellulosicdiaper product. This hydrolysis experiment was performed using 0.4 mg ofthe standard and the fermentation product per 100 mg of substrate (thecellulosic diaper). The experiment was run at 50° C. over a period offive hours and the glucose concentration was measured, in duplicate, atvarious time intervals. This curve illustrates an increased rate ofhydrolysis for the product produced by the fermentation product derivedfrom the transformant having additional copies of bg11, compared to thestandard. It was also determined that the diaper derived fibers wereabout 14% insoluble in aqueous solution. Thus, the fermentation productobtained from the transformants or the transformants alone can be usedin compositions to help degrade by liquefaction a variety of celluloseproducts that add to overcrowded landfills.

Simultaneous saccharification and fermentation is a process wherebycellulose present in biomass is converted to glucose and, at the sametime and in the same reactor, yeast strains convert the glucose intoethanol. Yeast strains that are known for use in this type of processinclude B. clausenii, S. cerevisiae, Cellulolyticus acidothermo-philium, C. brassicae, C. lustinaniae, S. uvarum, Schizosaccharomycespombe and the like. Ethanol from this process can be further used as anoctane enhancer or directly as a fuel in lieu of gasoline which isadvantageous because ethanol as a fuel source is more environmentallyfriendly than petroleum derived products. It is known that the use ofethanol will improve air quality and possibly reduce local ozone levelsand smog. Moreover, utilization of ethanol in lieu of gasoline can be ofstrategic importance in buffering the impact of sudden shifts innon-renewable energy and petro-chemical supplies.

Ethanol can be produced via saccharification and fermentation processesfrom cellulosic biomass such as trees, herbaceous plants, municipalsolid waste and agricultural and forestry residues. However, one majorproblem encountered in this process is the lack of β-glucosidase in thesystem to convert cellobiose to glucose. It is known that cellobioseacts as an inhibitor of cellobiohydrolases and endoglucanases andthereby reduces the rate of hydrolysis for the entire cellulase system.Therefore, the use of increased β-glucosidase activity to quicklyconvert cellobiose into glucose would greatly enhance the production ofethanol. To illustrate this point, cytolase 123 and the fermentationproduct produced by the transformants (normalized to cytolase on a totalprotein basis) according to the present invention under fermentationconditions were compared for their ability to hydrolyze crude paperfractions composed of 50-60% cellulosics from a fiber fraction (RDF) ofmunicipal solid waste (MSW). Such suspensions were in 50 mM sodiumacetate buffer, pH 4.8 to 5.0, and equilibrated at 30° C. The flaskswere then dosed with 4% Saccharomyces cerevisiae and sampledperiodically to 80 hours. The ethanol production yield was thenmeasured. The following Table II illustrates that increased ethanolproduction is possible using the increased β-glucosidase preparationfrom the present invention using municipal solid waste preparations asthe cellulosic source.

                  TABLE II                                                        ______________________________________                                        Dosage         Grams/Liter Ethanol                                            mg Protein     Cytolase                                                       gram cellulose 123      High β-Glu Prep                                  ______________________________________                                        10             2.1      5.0                                                   20             5.3      7.2                                                   30             6.9      8.8                                                   40             8.0      9.3                                                   50             8.5      9.3                                                   60             8.5      9.3                                                   ______________________________________                                    

From Table II it can be clearly seen that the enhance β-glucosidasepreparation prepared according to the present invention enhances theproduction of ethanol compared to a cytolase 123 control, especially atthe lower protein concentrations.

In yet another embodiment of this invention, the deletion of the bg11gene from T. reesei strains would be particularly useful in preparingcellulase compositions for use in detergents and in isolatingcellooligosaccharides (e.g., cellobiose).

The cellulase enzymes have been used in a variety of detergentcompositions to enzymatically soften clothes and to provide colorrestoration. However, it is known in this art that use of cellulaseenzymes can impart degradation of the cellulose fibers in clothes. Onepossibility to decrease the degradation effect is to produce a detergentthat does not contain β-glucosidase. Thus, the deletion of this proteinwould effect the cellulase system to inhibit the other components viaaccumulation of cellobiose. The modified microorganisms of thisinvention are particularly suitable for preparing such compositionsbecause the bg11 gene can be deleted leaving the remaining CBH and EGcomponents thereby resulting in color restoration and improved softeningbenefits in the composition without degradative effects.

The detergent compositions of this invention may employ, besides thecellulase composition (deleted in β-glucosidase), a surfactant,including anionic, non-ionic and ampholytic surfactants, a hydrolase,building agents, bleaching agents, bluing agents and fluorescent dyes,caking inhibitors, solubilizers, cationic surfactants and the like. Allof these components are known in the detergent art. For a more thoroughdiscussion, see U.S. Application Ser. No. 07/593,919 entitled"Trichoderma reesei Containing Deleted Cellulase Genes and DetergentCompositions Containing Cellulases Derived Therefrom", and U.S. Ser. No.07/770,049, filed Oct. 4, 1991 and entitled "Trichoderma reeseiContaining Deleted and/or enriched Cellulase and other enzyme Genes andCellulase Compositions Derived Therefrom" both of which are incorporatedherein by reference in their entirety.

In yet another embodiment, the detergent compositions can also containenhanced levels of β-glucosidase or altered β-glucosidase. In thisregard, it really depends upon the type of product one desires to use indetergent compositions to give the appropriate effects.

Preferably the cellulase compositions are employed from about 0.00005weight percent to about 5 weight percent relative to the total detergentcomposition. More preferably, the cellulase compositions are employedfrom about 0.01 weight percent to about 5 weight percent relative to thetotal detergent composition and even more preferably, from about 0.05 toabout 2 weight percent relative to the total detergent composition.

Deletion of the bg11 gene would also provide accumulation ofcellooligosaccharides (e.g., cellobiose) in cellolosic solutions treatedwith cellulase system, which can be purified therefrom. In this regard,the present invention presents the possibility to isolatecellooligosaccharides employing microorganisms in an easy and effectivemanner.

Cellooligosaccharides are useful in assaying cellulase enzymes forenzymatic activity and are also useful in the synthesis of ethanol andglucose. Moreover, it is contemplated that such oligosaccharides wouldalso be useful as food additives, chemical intermediates, etc.

Heretofore, the use of cellulase containing β-glucosidase to preparecellooligosaccharides required the deactivation of β-glucosidase byadjusting the pH of the solution to less than about 4 and generally toaround 3.8. At this pH, the β-glucosidase is generally inactivated.However, at this pH, the other enzyme components of cellulase aregenerally less active as compared to their optimum pHs and, accordingly,such a reduction of pH to inactivate the β-glucosidase necessarilyresults in a less efficient process.

On the other hand, the use of cellulase compositions free ofβ-glucosidase as per this invention provides a facile means forpreparing cellooligosaccharides at a pH of from about 4.5 to about 8;preferably at a pH of from about 4.5 to about 6 and most preferably atthe pH optimum for the cellulase composition employed. In thisembodiment, the invention is directed to a process for producingcellooligosaccharides which comprises contacting cellulose containingmaterials (i.e., materials containing at least 20% cellulose andpreferably at least 50% cellulose) with a cellulase composition free ofβ-glucosidase at a pH of from about 4.5 to about 8. Additionally,cellulase compositions containing reduced amounts of β-glucosidase canbe obtained by mixing the cellulase produced by a β-glucosidicfilamentous fungi and the cellulase produced by a β-glucosidicfilamentous fungi which has been modified to be incapable of producingβ-glucosidase.

Moreover, the present invention also contemplates the use of theβ-glucosidase nucleotide sequence of T. reesei to design various probesfor the identification of the extracellular β-glucosidase gene in otherfilamentous fungi. In this regard, the entire nucleotide sequence of thebg11 gene can be used or a portion thereof to identify and clone out theequivalent genes from other filamentous fungi. The sources offilamentous fungi include those fungi from the genus Trichoderma,Aspergillus, Neurospora, Humicola, Penicillium and the like. Moreparticularly, the preferred species include Trichoderma reesei,Trichoderma viridae, Aspergillus niger, Aspergillus oryzae, Neurosporacrassa, Humicola arisea, Humicola insolens, Penicillium pinophilum,Penicillium oxalicum, Aspergillus phoenicis, Trichoderma koningii andthe like. Due to the species homology of the bg11 gene, filamentousfungi equivalent genes are easily identified and cloned. Indicative ofthis are FIGS. 10A and 10B which illustrate autoradiograph of A.nidulans and N. crassa (FIG. 10A) and H. grisea (FIG. 10B) DNA digestedwith Hind III and Eco RI and further were blotted and probed with a 2labeled Hind III 6.0 kb bg11 DNA fragment containing the bg11 gene of T.reesei. These autoradiographs clearly illustrate that a DNA fragmentcontaining the bg11 gene of T. reesei can be used to identify theextracellular bg11 gene in other fungi.

Thus the bg11 gene of other filamentous fungi may be cloned by themethods outlined above using the P³² labelled T. reesei bg11 gene as aprobe. Once the genes of other filamentous fungi are cloned, they can beused to transform the filamentous fungi from which the gene was derivedor other filamentous fungi to overproduce β-glucosidase by the methodsdescribed above. Alternatively, the cloned bg11 genes from the otherfilamentous fungi can be used by the methods described above to deleteor disrupt the bg11 gene in the genome of the filamentous fungi fromwhich the bg11 gene was originally cloned.

In order to further illustrate the present invention and advantagesthereof, the following specific examples are given, it being understoodthat the same are intended only as illustrative and in nowiselimitative.

EXAMPLE 1 Isolation of Total RNA from Trichoderma reesei

A Trichoderma reesei culture which over produces cellulases wasspecifically induced for cellulase using sophorose, a β,1-2 diglucosideas described by Gritzali, 1977. The starting strain of Trichodermareesei is a cellulase over-production strain (RL-P37) developed bymutagenesis by the methods described by Sheir-Neiss, G. andMontenecourt, B. S., Appl. Microbiol. Biotechnol., Vol. 20 (1984) pp.46-53. A mycelial inoculum of T. reesei, from growth on potato dextroseagar (Difco), was added into 50 ml of Trichoderma basal mediumcontaining 1.40 grams/liter (NH₄)₂ SO₄, 2.0 grams/liter KH₂ PO₄, 0.30grams/liter MgSO₄, 0.30 grams/liter urea, 7.50 grams/liter BactoPeptone,5.0 ml/liter, 10% Tween--80, 1.0 ml/liter trace elements-EFG, pH 5.4,which was filtered through a 0.2 micron filter in a 250 ml baffledflask. This culture was incubated at 30° C. for 48 hours with vigorousaeration. Five milliliter aliquots were taken from the culture and addedto 25 ml of fresh basal medium in seven 250 ml flasks. These weresubsequently grown for 24 hours at 30° C. All cultures were centrifugedin a benchtop clinical centrifuge at 2400× for 10 minutes. The mycelialpellets were washed three times in 50 mls of 17 mM KHPO₄ buffer (pH6.0). Lastly, the mycelia were suspended in six flasks containing 50 mlof 17 mM KHPO₄ buffer with the addition of 1 mM sophorose and a controlflask containing no sophorose. The flasks were incubated for 18 hours at30° C. prior to harvesting by filtration through Mira-cloth(Calbiochem). The excess medium was then squeezed out and the mycelialmat was placed directly into liquid nitrogen and may be stored at -70°C. for up to one month. The frozen hyphae were then ground in anelectric coffee grinder that was prechilled with a few chips of dry iceuntil a fine powder was obtained. The powder was then added to about 20ml of an extraction buffer containing 9.6 grams of p-aminosalicylic aciddissolved in 80 ml of DEP-treated water, 1.6 grams oftriisopropylnaphthalene sulfonic acid dissolved in 80 ml of DEP-treatedwater, 24.2 grams Tris-HCl, 14.6 grams NaCl, 19.09 grams EDTA, which wasdiluted to 200 ml total volume with DEP-treated water and the pH wasadjusted to 8.5 with NaOH. After addition of the extraction buffer, 0.5volumes of TE-saturated phenol was also added thereto, and theextraction mixture was placed on ice. One quarter volume of chloroformwas then added to the extraction mixture, and the mixture was shaken fortwo minutes. The phases were then separated by centrifugation at 2500rpm. The aqueous phase was removed and placed in a centrifuge tube,which contained a few drops of phenol in the bottom of said tube. Thetube was placed on ice. The organic phase was then reextracted with 2.0ml of extraction buffer and placed in a 68° C. water bath for 5 minutesto release the RNA trapped in polysomes and at the interface of theextraction mixture. The extracted mixture was then centrifuged, and theaqueous phase removed and pooled with the other aqueous fraction.

The entire aqueous fractions were then extracted with phenol-chloroform(1:1 v/v) for 4 to 5 times until there was no longer any protein seenvisually at the interface. Then 0.1 volume of 3 M sodium acetate, pH 5.2(made with DEP water and autoclaved) and 2.5 volumes of 95% was added tothe organic extracts, and the extracts were frozen at -20° C. for 2 to 3hours. Alternatively, the RNA was precipitated using 2 M lithiumacetate. The RNA was then pelleted by centrifugation at 12,000 rpm for20 minutes. The pelleted RNA was then resuspended in DEP-water with anRNase inhibitor to a final concentration of 1 unit per pl. To determinewhether the genes encoding the enzymes were being induced, total RNA wasanalyzed.

Analysis of Total RNA Preparation

To confirm whether the genes encoding the enzymes of the cellulasecomplex were being induced, total RNA was analyzed by Northern blottingas described by Sambrook et al, supra using a 2 fragment of the T.reesei cbh2 gene as a probe. The cbh2 clone was isolated using themethods described by Chen et al in "Nucleotide Sequence and DeducedPrimary Structure of Cellobio-hydrolase II from Trichoderma reesei",Biotechnology, Vol. 5 (March 1987), incorporated herein by reference.Site directed mutagenesis (Sambrook et al., supra) was performed on thecbh2 clone and a Bgl II site was placed at the exact 5' end of theopening reading frame and an Nhe I site at the exact 3' end. The BglII/Nhe I coding sequence was then cloned into a pUC218 phagemid. For useas a probe, the cbh2 fragment was digested with Bgl II/Nhe I andisolated by gel electrophoresis. The results indicated that the level ofcbh2 specific mRNA reached a peak at 14-18 hours post induction. Thetotal RNA from 14, 18 and 22 hours was then pooled.

EXAMPLE 2 Purification of Polvadenylated mRNA

mRNA was then isolated from the pooled fraction of total RNA set forthabove using oligo (dT) cellulose chromatography. Oligo(dT) cellulose(type 3 from Collaborative Research, Lexington, Mass.) is firstequilibrated with oligo(dT) binding buffer containing 0.01 M Tris-HCl,pH 7.5, 0.5 M NaCl, and 1 mM EDTA, then aliquots of 25-300 mg were addedto 1.5 ml microfuge tubes. RNA dissolved in 1 ml of binding buffer wasadded and allowed to bind for 15 min. with gentle shaking. Thesuspensions were centrifuged at 1500 g for 3-5 min., washed 3-5 timeswith 1 ml of binding buffer, and then washed 3 times with 400 μl ofelution buffer containing 0.01 M Tris-HCl, pH 7.5, and 1 mM EDTA. Theeluates were pooled, readjusted to 0.5 M NaCl, rebound, and reelutedwith three washes of elution buffer. The final three elution bufferwashes were pooled and mRNA was recovered by ethanol precipitation.

Analysis of Total RNA and polyadenylated mRNA

Total RNA and the polyadenylated RNA were fractionated on 1%formaldehyde-agarose gels using 10 μg of RNA for each lane, blotted toNytran^(R) membranes and analyzed by the Northern blot method describedby Thomas in "Hybridization of denatured RNA and Small DNA fragmentstransferred to Nitrocellulose", Proc. Natl. Acad. Sci. USA, Vol. 77(1980), pp. 5201-5205.

Briefly, this procedure involves denaturing RNA (up to 10 μg/8 μlreaction) by incubation in 1 M glyoxal/50% (vol/vol) Me₂ SO/10 mM sodiumphosphate buffer, pH 7.0 at 50° C. for 1 hr. The reaction mixture wascooled on ice and 2 μl of sample buffer containing 50% (vol/vol)glycerol, 10 mM sodium phosphate buffer at 7.0 and bromophenol blue wasadded. The samples were electro-phoresed on horizontal 1%formaldehyde-agarose gels in 10 mM phosphate buffer, pH 7.0 at 90 v for6 hours.

The glyoxylated RNA was transferred from agarose gels to nitrocelluloseby using 3 M NaCl/0.3 M trisodium citrate (20X NaCl/cit). Afterelectrophoresis, the gel was placed over two sheets of Whatman 3 MMpaper which was saturated with 20X NaCl/cit. Nitran^(R) membrane waswetted with water, equilibrated with 20X NaCl/cit and laid over the gel.The gel was then covered with two sheets of Whatman 3 MM paper and a 5to 7 cm layer of paper towels, a glass plate and a weight. Transfer ofthe RNA was completed in 12-15 hours. The blots were then dried under alamp and baked in a vacuum for over 2 hrs. at 80° C.

The membranes were probed with a cbh2 probe to verify that thepolyadenylated MRNA pool contained cbh2 mRNA and by inference the genesencoding the enzymes of the cellulase complex were indeed induced.

EXAMPLE 3 Synthesis of cDNA

A. First Strand Synthesis

Synthesis of cDNA was performed using the BRL cDNA Synthesis System^(R)(Bethesda Research Laboratories, Md.) according to the instructions ofthe manufacturer. To a sterile, DEPC-treated tube in ice was added 10 μlof 5X First Strand Buffer containing 250 mM Tris-HCl, pH 8.3, 375 mMKCl, 15 mM MgCl₂, 50 mM DTT, 2.5 μl 10 mM dNTP Mix (10 mM DATP, 10 mMdCTP, 10 mM dGTP, 10 mM dTTP), 5 μl Oligo (dT),₁₂₋₁₈ (0.5) mg/ml), 10 μlof mRNA at 0.5 mg/ml and 20 μl diethylpirocarbmate(DEPC)-treated waterto create a final composition containing 50 mM Tris-HCl (pH 8.3), 75 mMKCl, 3 mM MgCl₂, 10 mM dithiothreitol, 500 μM each DATP, dCTP, dGTP anddTTP, 50 μg/ml Oligo (dT)₁₂₋₁₈, 100 μg/ml polyadenylated RNA and 10,000U/ml cloned M-MLV reverse transcriptase. A control run was also runsimultaneously using 10 μl of a 2.3 kb control RNA (0.5 mg/ml) in lieuof the mRNA.

The reaction was initiated by adding 2.5 μl of Molony murine leukemiavirus (M-MLV) reverse transcriptase (100 Units/μl) to the MRNA tube andthe control RNA. The samples were mixed. All reaction tubes wereincubated at 37° C. for one hour and then placed on ice.

A small aliquot from the reaction mixture was run on a gel to confirmits presence and quantity. The yield obtained was about 2-6 μg.

B. Second Strand Synthesis

To the control tube on ice after first strand synthesis was added 230.6μl DEPC-treated water, 6 μl 10 mM dNTP mix, 32 μl 10X second strandbuffer containing 188 mM Tris-HCl, pH 8.3, 906 mM KCl, 100 mM (NH₄)₂SO₄, 46 MM MgCl₂, 37.5 mM dithiothreitol, 1.5 mM NAD, 8 μl E. coli DNAPolymerase I (10 μ/μl), 1.4 μl E. coli RNase H and 1 μl E. coli DNAligase (100 units).

To the first strand synthesis of the sample was added on ice 289.5 μl ofDEPC-treated water, 7.5 μl 10 mM dNTP mix, 40 μl 10X second strandbuffer, 10 μl E. coli DNA Polymerase I, 1.75 μl E. coli RNaseH and 1.25E. coli DNA ligase, to create a final composition containing 25 mMTris-HCl (pH 8.3), 100 mM KCl, 10 mM (NH₄)₂ SO₄, 5 mM MgCl₂, 250 μM eachdATP, dCTP, dGTP, dTTP, 0.15 mM NAD, 5 mM Dithiothreitol, 250 U/ml DNAPolymerase I, 8.5 U/ml RNase H, and 30 U/ml DNA Ligase. Both the controltube and the sample tube were vortexed gently and incubated for 2 hoursat 16° C. After incubation, both tubes were placed on ice.

The sample tube was then extracted with 415 μl of phenol and ethanolprecipitated. The pellet was dissolved in 200 μl of sterile TE buffer(10 mM Tris-HCl pH 7.5, 1 mM Na₂ EDTA) and reprecipitated from 7.5 Mammonium acetate with ethanol.

An aliquot of the sample was further analyzed by gel electrophoresis tocheck for purity. The yield of the synthesis was about 4.0 μg.

The remaining control sample was further extracted with phenol andethanol precipitated as described above for the sample. After dissolvingthe pellet in 200 μl of sterile TE buffer, reprecipitating the samplefrom ammonium acetate with ethanol, and redissolving the dry pellet in20 μl of sterile TE buffer, 2 μl of the solution was then furtheranalyzed by gel electrophoresis to check for purity.

EXAMPLE 4 Amplification of bg11 cDNA Sequences

Amplification of the cDNA fragments encoding a portion of the T. reeseiβ-glucosidase gene, bg11, was performed using the polymerase chainreaction (PCR) method with Taq^(R) polymerase and a Perkin Elmer CetusThermal Cycler^(R).

The reaction mixture was formed by mixing 76 μl deionized water, 10 μlof a 10X mixture of buffer containing 166 mM (NH₄)₂ SO₄, 670 mMTris-HCl, pH 8.8, 67 mM MgCl₂, 67 μm EDTA, 10 mM β-mercaptoethanol, 10μl dimethylsulfoxide and 1.7 mg/ml BSA diluted to a total volume of 1.0ml with deionized water, 8 μl of 2 dNTPs (each), 1 μl 5' oligonucleotideprimer, 1 μl 3' internal oligonucleotide primer, 1.0 μg cDNA diluted in3 μl deionized water, and 1 μg Taq^(R) polymerase.

The amplification method consists of an initial denaturing cycle at 95°C. for 10 minutes, followed by a two minute annealing step at 50° C. anda 10 minute polymerization cycle at 65° C., for an additional 30 cycles.

A. Oligonucleotide Primers

The oligonucleotide primers used to amplify the cDNA fragment encodingthe T. reesei bg11 gene were designed based on the degeneracy of thegenetic code for the selected amino acids for an N terminal region ofthe bg11 gene and an internal oligonucleotide. The 5' oligonucleotideprimer consisted of the sequence:

5' GCI GTI CCT CCT GCI GG 3'(SEQ ID NO:3)

wherein I=inosine.

The internal 3' oligonucleotide primer consisted of a pool of 16×21oligonucleotides. This pool was based on various derivations of thefollowing sequences:

5' GTT G/ATT ICC G/ATT G/AAA G/ATC TGT 3'(SEQ ID NO:4)

EXAMPLE 5 Subcloning of PCR Generated Fragments

Ninety μl of each reaction mix was fractionated on 4% polyacrylamidegels in 1X TBE, the major band was excised and eluted from the gel sliceas described by Sambrook et al, supra. The eluted DNA fragment wasprecipitated in ethanol and resuspended in 15 μl of TE buffer (10 mMTris, 1 mM EDTA). Each 1-2 μg DNA fragment was then treated with 0.5 mMATP and T₄ polynucleotide kinase to phosphorylate the 5' end of eachfragment following by the procedures of Sambrook et al, supra. Bluntends were generated by adding 3 μl of 10X T₄ polynerase buffer (330 mMTris-acetate at pH 7.9, 660 mM potassium acetate, 100 mM magnesiumacetate, 1 μl of 2.5 mM dNTPS, 1 μl of T₄ DNA polymerase and 5 μl ofdistilled water). The blunt-end reaction mixture was then incubated at37° for 60 minutes. The reaction was stopped by addition of EDTA to afinal concentration of 1 mM EDTA and the sample was further heated for10 minutes at 65° C.

The blunt-end DNA fragments were then ligated with SmaI cleaved anddephosphorylated pUC218 which had been infected with M13X07 as describedby Sambrook et al, supra. The cloning vectors pUC218 and pUC219 werederived from pUC118 and pUC119 by insertion of the Bgl II, Cla I and XhoI polylinker as described by Korman et al in "Cloning, Characterization,and expression of two α-amylase genes from Aspergillus niger var.awamori", Current Genetics, Vol. 17, pp. 203-212, (1990).

The aforedescribed phagemid was then used to transform E. coli strainJM101 as described by Yarnisch et al in "Improved M13 phage cloningvectors and host strains: nucleotide sequence of the M13mp18 and pUC19Vectors" Gene, Vol. 1197, pp. 103-119 (1985).

EXAMPLE 6 Isolation of cDNA Subcloned Fragment

The transformed strain was inoculated in 1.5 ml of 2YT broth in a tubewhich had been previously inoculated with 15 μl of saturated E. coliJM101. The culture was grown for 8 hours under shaking at 37° C.

The culture mixture was then spun at 6000 rpm for 5 minutes, and thesupernatant was poured off into another tube. To the supernatant 300 μlof 2.5 M NaCl, 20% PEG was added, and the solution was mixed. Themixture was then incubated at room temperature for 15 minutes.

The solution was then spun for 5 minutes in a microfuge, and thesupernatant was aspirated off. The solution was vortexed once again, andthe supernatant was further aspirated off. 100 μl of equilibrated phenolwas added to the tube, and the tube was vortexed. 100 μl of chloroformwas added, and once again the tube was vortexed. The tube was heated at55° C. for 5 minutes, mixed, and microfuged an additional 5 minutes. 160μl of the supernatant was then pipetted off and transferred to a cleartube. 20 μl of iN NaOAC, pH 4.5, and 400 μl of 95% ETOH were added tothe supernatant, and the solution was mixed and frozen on dry ice for 5minutes. The tube was then spun for an additional 15 minutes, and thesupernatant was aspirated off.

1000 μl of 70% ethanol was added to the tube, and the tube was spun foran additional 2 minutes and reaspirated. The mixture was spun once moreunder vacuum for 4 minutes, and the pellet was resuspended in 15 pl TEbuffer.

EXAMPLE 7 Determination of the Nucleotide Sequence of 700 bp cDNAfragment

The nucleotide sequence of the 700 bp cDNA fragment was determined usingthe dideoxy DNA sequencing method described by Sanger et al, "DNASequencing with chain terminating inhibitors", Proc. Natl. Acad. Sci.U.S.A., Vol. 74 (1977), p. 5463, using the Sequenase^(R) reagent kit(U.S. Biochemicals).

EXAMPLE 8 Analysis of bg11 gene

A. Sequence Analysis

Nucleotide sequencing was done by the dideoxy chain termination methodof Sanger et al (1977) using the Sequenase^(R) reagent kit (U.S.Biochemicals).

B. Amino Acid Sequencing

A 2.5-nmol sample of the reduced and carboxy-methylated β-glucosidasepreparation purified (per Chirico and Brown, European Journal ofBiochem., Vol. 165, pp. 333 et seq.) was subjected to N-terminalsequencing on a proprietary multiphase sequencer.

To a sample of β-glucosidase, Endo-Lys C protease was added to 1% of thetotal protein and the mixture incubated for 1 hour at 37° C. or theprotein sample was subject to cyanogen bromide treatment. An equalvolume of HPLC solution A (0.05% TEA/0.05% TFA in water) was added tostop the reaction. The resulting CNBr and Endo-Lys C fragments wereseparated by chromatography on a Brownlee C-4 column using a lineargradient of 0-100% HPLC solution B (0.05% TEA/0.05% TFA in n-propanol)at a rate of 1% per minute. Several peaks were collected for amino acidsequencing and the data are denoted in FIG. 1.

EXAMPLE 9 Identification of bg11 gene from T. reesei

The 700 bp bg11 cDNA fragment was then labelled with ³² P using methodsdescribed by Sambrook et al, supra.

Genomic DNA from T. reesei was prepared by filtering a 24-36 hourculture of T. reesei through Miracloth and freezing the mycelia obtainedfrom the culture medium. The frozen mycelia were then ground into a finepowder and 22 mls of TE, and 4 mls of 20% SDS were added to the powderedmycelia and mixed. 10 ml of phenol and chloroform was added to themixture prior to centrifugation and removal of the aqueous phase. 200 μlof 5 mg/ml proteinase K was added to the organic extract, and themixture was incubated for 20 minutes at 55° C. The DNA was then furtherextracted by methods known in the art using chloroform/phenol extractionfollowed by ethanol precipitation. The isolated DNA was then treatedwith 1 μg of heated ribonuclease A (100° C. for 15 minutes) per 20 μg ofgenomic DNA in TE buffer at 37° C. for 30 minutes, then cooled to roomtemperature. The genomic DNA from T. reesei was then cut singly or incombination with a variety of restriction enzymes such as Eco RI, HindIII and the like, Southern blotted and hybridized with the P³² labelled700 bp cDNA fragment of the bg11 gene as a probe. From this analysis itwas determined that Hind III was the restriction enzyme of choice usedto locate the β-glucosidase gene.

10 to 20 units of Hind III per milligram of genomic DNA was added to theDNA and then the DNA was extracted with phenol-chloroform to removeprotein. The treated DNA was then alcohol precipitation and resuspendedto 2 grams/liter in TE buffer.

4 μl samples from the Hind III digestion of genomic DNA were loaded on a1% agarose gel and fractionated electrophoretically. The gel was thenSouthern blotted and probed with the P³² 700 bp cDNA probe. A 6.0 kbband was identified on the Southern blot of Hind III digested genomicDNA from T. reesei.

The remaining Hind III genomic DNA was then subjected to a preparativegel electrophoresis and fragments ranging from 5 kb to 7 kb were thenelectroeluted from the agarose gel and cloned into Hind III digestedpUC218. The resulting plasmids were used to transform E. coli JM101 tocreate a library. Then the library was screened by colony hybridizationusing P³² labelled 700 bp bg11 cDNA as a probe to identify thosecolonies containing DNA coding for the bg11 gene.

The positive colonies from the transformation were then picked and theDNA isolated therefrom by phenol:chloroform extraction and ethanolprecipitation, described by Sambrook et al, supra.

The isolated DNA from the positive colonies was digested both singly andin various combinations with the following restriction enzymes: HindIII, Eco RI, Sst 1, Kpn I, Bam HI, Xho 1, Bgl II, Cla I, Xba I, Sal I,Pst I, Sph I, Bal I, and Pvu II. The digestions were subjected toagarose gel electrophoresis, and the resultant banding pattern was usedto construct a restriction map of the cloned 6.0 kb genomic DNA. Thesame agarose gel was Southern blotted and probed with the P³² labelled700 bp bg11 cDNA to identify which genomic restriction fragments sharedhomology with the bg11 cDNA. The mapping experiments confirmed that theentire bg11 gene is contained on the genomic Hind III clone. Pvu II andBal I restriction fragments which ranged in size from 600 bp to 1500 bphybridized with the 700 bp DNA bg11 clone and were chosen for subcloninginto pUC218 phagemid. After cloning these fragments into the phagemid,the Pvu II and Bal I subclones were then sequenced using the dideoxychain termination method of Sanger et al (1977). It was then determinedfrom this sequencing that the overlapping sequences of the subclonesaligned with a single contiguous sequence totaling 3033 bp within whichthe nucleotide sequence was determined on both strands.

EXAMPLE 10 Construction of pSASβ-glu

The starting vector for the construction of pSASβ-glu was the plasmidpSAS. pSAS was constructed in the following way. pUC100 (a commerciallyavailable plasmid vector) was digested with the restriction enzyme SmaIand the 5' phosphate groups subsequently removed by digestion with calfintestinal alkaline phosphatase. The linear vector fragment was purifiedfrom undigested vector and protein by agarose gel electrophoresisfollowed by isolation of the linear vector DNA from the isolated gelslice by electro-elution. The amdS gene was isolated as a 2.4 kb SstIrestriction fragment following separation from the vector sequences(contained in--Hynes, M. J., Corrick, C. M., and King, J. A., "Isolationof genomic clones containing the amdS gene of Aspergillus nidulans andtheir use in the analysis of structural and regulatory mutations", Mol.Cell. Biol., Vol. 3 (1983), pp. 1430-1439). The 2.4 kb SstI amdSfragment and the 2.7 kb pUC100 vector fragment were then ligatedtogether (Sambrook et al., supra) and the ligation mix transformed andpropagated in the E. coli host strain, JM101.

pSASβ-glu was constructed by digesting pSAS with the restriction enzymeHind III, and purifying the linear fragment as described above. Intothis Hind III treated pSAS vector fragment was ligated a 6.0 kb Hind IIIfragment of T. reesei genomic DNA that contained all of the codingregion of the bg11 gene along with sequences necessary for the genestranscription and translation.

EXAMPLE 11 Preparation of BGL1 Deletion yector

The gene replacement vector pUCΔβ-Glu A/R pyr, illustrated in FIG. 3B,was constructed by cloning a 6.0 kb genomic HindIII fragment, known tocontain the entire bg11 gene, into the polylinker of pUC218 which hadbeen cut with HindIII and the ends dephosphorylated with calf intestinalalkaline phosphatase. The coding region for the bg11 gene was thenremoved from this plasmid by digesting the plasmid with ApaI and EcoRVat unique ApaI and EcoRV restriction sites situated at the very 5' and3' end of the bg11 open reading frame and isolating the linear plasmidDNA. The restriction site ends were made blunt with T4 DNA polymerase.This plasmid was then ligated with an isolated 2412 bp Hind III/Bam HIrestriction fragment containing the pyrG gene from Aspergillus niger(Hartingsreldt et al., Mol. Gen. Genet. 206:71-75 (1987) in which therestriction ends were made blunt by treatment with T4 DNA polymerase tocreate pUCΔβGlu A/R pyr (FIG. 3B).

EXAMPLE 12 Isolation of Protoplasts

Mycelium was obtained by inoculating 100 ml of YEG (0.5% yeast extract,2% glucose) in a 500 ml flask with about 5×10⁷ T. reesei cells. Theflask was then incubated at 37° C. with shaking for about 16 hours. Themycelium was harvested by centrifugation at 2,750×g. The harvestedmycelium were further washed in 1.2 M sorbitol solution and resuspendedin 40 ml of Novozymt, which is the tradename for a multi-componentenzyme system containing 1,3-alpha-glucanase, 1,3-beta-glucanase,laminarinase, xylanase, chitinase and protease from Novo Biolabs,Danbury, Conn., solution containing 5 mg/ml Novozym^(R) 234; 5 mg/mlMgSO₄.7H₂ O; 0.5 mg/ml bovine serum albumin; 1.2 M sorbitol. Theprotoplasts were removed from cellular debris by filtration throughMiracloth (Calbiochem Corp.) and collected by centrifugation at 2,000×g.The protoplasts were washed three times in 1.2 M sorbitol and once in1.2 M sorbitol, 50 mM CaCl₂, centrifuged and resuspended. Theprotoplasts were finally resuspended at a density of 2×10⁸ protoplastsper ml of 1.2 M sorbitol, 50 mM CaCl₂.

EXAMPLE 13 Transformation of Fungal Protoplasts with pSASβ-glu

200 μl of the protoplast suspension prepared in Example 12 was added to20 μl (20 μg) of pSASβ-glu in TE buffer (10 mM Tris, pH 7.4; 1 mM EDTA)and 50 μl of a polyethylene glycol (PEG) solution containing 25% PEG4000, 0.6 M KC1 and 50 mM CaCl₂. This mixture was incubated on ice for20 minutes. After this incubation period 2.0 ml of the above-identifiedPEG solution was added thereto, the solution was further mixed andincubated at room temperature for 5 minutes. After this secondincubation, 4.0 ml of a solution containing 1.2 M sorbitol and 50 mMCaCl₂ was added thereto and this solution was further mixed. Theprotoplast solution was then immediately added to molten aliquot's ofVogels Medium N (3 grams sodium citrate, 5 grams KH₂ PO₄, 2 grams NH₄NO₃, 0.2 grams MgSO₄.7H₂ O, 0.1 gram CaCl₂.2H₂ O, 5 μg α-biotin, 5 mgcitric acid, 5 mg ZnSO₄.7H₂ O, 1 mg Fe(NH₄)₂.6H₂ O, 0.25 mg CuSO₄.5H₂ O,50 μg MnSO₄.4H containing an additional 1% glucose, 1.2 M sorbitol and1% agarose. The protoplast/medium mixture was then poured onto a solidmedium containing the same Vogel's medium as stated above containing inaddition acetamide as a nitrogen source. Since T. reesei does notcontain a functional equivalent to the amdS gene only transformants willgrow on this medium. These colonies were subsequently transferred andpurified on a solid Vogel's medium N containing as an additive, 1%glucose. The bg11 gene inserted transformant strain is calledA83pSASβGlu.

Stable transformants can be distinguished from unstable transformants bytheir faster growth rate and the formation of circular colonies with asmooth rather than ragged outline on solid culture medium. Additionally,in some cases, a further test of stability can be made by growing thetransformants on solid non-selective medium, harvesting the spores fromthis culture medium and determining the percentage of these spores whichwill subsequently germinate and grow on selective medium.

FIG. 6 is an autoradiograph of a Southern blot using the P³² labelled700 bp fragment as a probe, of the different transformants with enhancedcopies of the bg11 gene using genomic T. reesei from an overproducingstrain digested with Hind III as a control. This autoradiograph clearlyshows that the transformants contained enhanced amount of the bg11 genecompared with the control.

FIG. 4 is an autoradiograph of a Northerr blot of RNA isolated from oneof the transformed strains produced by the present invention followinginduction with soporose illustrating a corresponding increase in thelevels of bg11 message when compared to the parental strain of T.reesei.

Besides visual analysis of the transformants, quantitative analysis wasalso completed by cutting the appropriate bands out of the Nytran^(R)membrane and counting the radioactive label present therein in ascintillation counter. This experiment was performed to obtain a moreprecise estimate of the relative amounts of message as shown in TableIII below:

                  TABLE III                                                       ______________________________________                                                 Parental Trichoderma                                                                        Transformed Trichoderma                                CPM      reesei strain reesei strain                                          ______________________________________                                        CPM      14.4          25.4                                                   β-glu                                                                    message                                                                       CPM      227.1         95.2                                                   CBHII                                                                         CPM      0.0634        0.2668                                                 β-glu/                                                                   CBHII                                                                         ______________________________________                                    

Table III illustrates that the transformant produced by the process ofthe present invention has extra β-glucosidase MRNA and hence an increasein β-glucosidase enzyme resulting in an increase in specific activity.

EXAMPLE 14 Transformation of Fungal Protoplasts with pUCΔβGlu A/R pvr4

Mutants of T. reesei lacking the coding sequence for the extracellularβ-glucosidase gene, bglI, were obtained by a targeted gene replacementevent. pUCΔβGlu A/R pyr4 plasmid was digested with Hind III to obtain alinear HindIII fragment in which the bg11 coding sequences were replacedwith the pvrG gene from Asperaillus niger. Protoplasts were transformedwith the linear DNA fragment containing the bg11 flanking sequences andthe pyr4 by the methods of Examples 12 and 13. The deletiontransformants were called Δ12 and Δ36. After transformation, theprotoplast solution was then added to molten aliquots of Vogel's MediumN containing an additional 1% glucose, 1.2 M sorbitol and 1% agarose.The protoplast/mdium mixture was then pourred into a solid mediumcontaining the same Vogel's medium N. No uridine was present in themedium and therefore only transformed colonies were able to grow as aresult of complementation of the pvr4 mutation of the T. reesei strainRL-P37 by the wild type pyr4 gene inserted in the DNA fragment. Stabletransformants were then selected by the method recited in Example 13.

EXAMPLE 15 Analysis of the Transformants

The transformants were analyzed for the presence or absence of the bg11gene using the 700 bp cDNA probe recited above. The transformants weredigested using HindIII. Total genomic DNA from selected transformantswas digested with HindIII restriction enzyme, run on a 1% agarose gel,transferred to NitranR membrane and probed with a 2 labelled 700 bp cDNArectied above and visualized by autoradiography on X-ray film. Theresults of this analysis are set forth in FIG. 5A illustrate that thetransformants (Δ12 and Δ36) did not contain a band corresponding to thebg11 gene whereas the wild type strain (RL-P37, i.e., P-37) did.

mRNA isolated from the transformants of Example 14 and analyzed on aNorthern blot, as in Example 2. As indicated in FIG. 5B, Northern blotanalysis using the P³² labelled 2.2 Kb ApaI/EcoRV bg11 probe indicatedthat bg11 specific mRNA was present in T. resei RL-P37 pyrG69 and isabsent in the transformants Δ12 and Δ36.

Protein was recovered as per Example 8 above and then analyzed for thepresence of β-glucosidase by use of polyclonal antibodies (from rabbitschallenged with pure β-glucosidase) tagged with horseradish peroxidaseto permit detection. The antibodies were used to identify pureβ-glucosidase (100 ng--Column A; 1000 ng--Column B); cellulase producedfrom wild type T. reesei (Column C); and from cellulase produced by a T.reesei strain genetically engineered to delete the β-glucoidase gene(Column D). The results of this analysis are set forth in FIG. 5C andshow that only Column D did not contain β-glucosidase.

While the invention has been described in terms of various preferredembodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the scope thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope of thefollowing claims, including equivalents thereof.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 4                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3033 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                   #reesei   (A) ORGANISM: Trichoderma                                           -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: join(311..37 - #5, 446..2205, 2270..2679)             -     (ix) FEATURE:                                                                     (A) NAME/KEY: intron                                                          (B) LOCATION: 376..445                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: intron                                                          (B) LOCATION: 2206..2269                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - TGGCCACAGA GGGAGAGTTC GCGCTACCGC TTGGTCGAGG AAATGATCGC CC - #ACGGCCTC         60                                                                          - AAATCGTAAA TCTCGGTGTG GGTAGGAGTG CAACGATGGG ATTTGGCCGC AA - #TGCTGCCG        120                                                                          - AGCCCGAGTG TTTCTGCAAC GTTATCCAGG AGATTTGCGC TTGCCCAAGA GG - #GAGTTGAC        180                                                                          - GGGGAGAGTC CCAACTGGTT CCTTCAGTAA CGCCACCCTG GCAGACTATA TA - #ACTTGTGG        240                                                                          - ACAAGACTCT GCTTTGTTGA GTTCTTCCTA CCAGTCTTGA CCAAGACCAT TC - #TGTTGAGC        300                                                                          - CCAATCAGAA ATG CGT TAC CGA ACA GCA GCT GCG CT - #G GCA CTT GCC ACT           349                                                                          #Thr Ala Ala Ala Leu Ala Leu Ala Thr                                          #          10                                                                 - GGG CCC TTT GCT AGG GCA GAC AGT  CA  GT - #ATAGCTGG TCCATACTGG               395                                                                          Gly Pro Phe Ala Arg Ala Asp Ser  His                                          #     20                                                                      #TCA ACA     452TCCTGGA GACACCATGC TGACTCTTGA ATCAAGGTAG C                    #   Ser Thr                                                                   - TCG GGG GCC TCG GCT GAG GCA GTT GTA CCT CC - #T GCA GGG ACT CCA TGG          500                                                                          Ser Gly Ala Ser Ala Glu Ala Val Val Pro Pr - #o Ala Gly Thr Pro Trp           # 40                                                                          - GGA ACC GCG TAC GAC AAG GCG AAG GCC GCA TT - #G GCA AAG CTC AAT CTC          548                                                                          Gly Thr Ala Tyr Asp Lys Ala Lys Ala Ala Le - #u Ala Lys Leu Asn Leu           #                 55                                                          - CAA GAT AAG GTC GGC ATC GTG AGC GGT GTC GG - #C TGG AAC GGC GGT CCT          596                                                                          Gln Asp Lys Val Gly Ile Val Ser Gly Val Gl - #y Trp Asn Gly Gly Pro           #             70                                                              - TGC GTT GGA AAC ACA TCT CCG GCC TCC AAG AT - #C AGC TAT CCA TCG CTA          644                                                                          Cys Val Gly Asn Thr Ser Pro Ala Ser Lys Il - #e Ser Tyr Pro Ser Leu           #         85                                                                  - TGC CTT CAA GAC GGA CCC CTC GGT GTT CGA TA - #C TCG ACA GGC AGC ACA          692                                                                          Cys Leu Gln Asp Gly Pro Leu Gly Val Arg Ty - #r Ser Thr Gly Ser Thr           #    100                                                                      - GCC TTT ACG CCG GGC GTT CAA GCG GCC TCG AC - #G TGG GAT GTC AAT TTG          740                                                                          Ala Phe Thr Pro Gly Val Gln Ala Ala Ser Th - #r Trp Asp Val Asn Leu           105                 1 - #10                 1 - #15                 1 -       #20                                                                           - ATC CGC GAA CGT GGA CAG TTC ATC GGT GAG GA - #G GTG AAG GCC TCG GGG          788                                                                          Ile Arg Glu Arg Gly Gln Phe Ile Gly Glu Gl - #u Val Lys Ala Ser Gly           #               135                                                           - ATT CAT GTC ATA CTT GGT CCT GTG GCT GGG CC - #G CTG GGA AAG ACT CCG          836                                                                          Ile His Val Ile Leu Gly Pro Val Ala Gly Pr - #o Leu Gly Lys Thr Pro           #           150                                                               - CAG GGC GGT CGC AAC TGG GAG GGC TTC GGT GT - #C GAT CCA TAT CTC ACG          884                                                                          Gln Gly Gly Arg Asn Trp Glu Gly Phe Gly Va - #l Asp Pro Tyr Leu Thr           #       165                                                                   - GGC ATT GCC ATG GGT CAA ACC ATC AAC GGC AT - #C CAG TCG GTA GGC GTG          932                                                                          Gly Ile Ala Met Gly Gln Thr Ile Asn Gly Il - #e Gln Ser Val Gly Val           #   180                                                                       - CAG GCG ACA GCG AAG CAC TAT ATC CTC AAC GA - #G CAG GAG CTC AAT CGA          980                                                                          Gln Ala Thr Ala Lys His Tyr Ile Leu Asn Gl - #u Gln Glu Leu Asn Arg           185                 1 - #90                 1 - #95                 2 -       #00                                                                           - GAA ACC ATT TCG AGC AAC CCA GAT GAC CGA AC - #T CTC CAT GAG CTG TAT         1028                                                                          Glu Thr Ile Ser Ser Asn Pro Asp Asp Arg Th - #r Leu His Glu Leu Tyr           #               215                                                           - ACT TGG CCA TTT GCC GAC GCG GTT CAG GCC AA - #T GTC GCT TCT GTC ATG         1076                                                                          Thr Trp Pro Phe Ala Asp Ala Val Gln Ala As - #n Val Ala Ser Val Met           #           230                                                               - TGC TCG TAC AAC AAG GTC AAT ACC ACC TGG GC - #C TGC GAG GAT CAG TAC         1124                                                                          Cys Ser Tyr Asn Lys Val Asn Thr Thr Trp Al - #a Cys Glu Asp Gln Tyr           #       245                                                                   - ACG CTG CAG ACT GTG CTG AAA GAC CAG CTG GG - #G TTC CCA GGC TAT GTC         1172                                                                          Thr Leu Gln Thr Val Leu Lys Asp Gln Leu Gl - #y Phe Pro Gly Tyr Val           #   260                                                                       - ATG ACG GAC TGG AAC GCA CAG CAC ACG ACT GT - #C CAA AGC GCG AAT TCT         1220                                                                          Met Thr Asp Trp Asn Ala Gln His Thr Thr Va - #l Gln Ser Ala Asn Ser           265                 2 - #70                 2 - #75                 2 -       #80                                                                           - GGG CTT GAC ATG TCA ATG CCT GGC ACA GAC TT - #C AAC GGT AAC AAT CGG         1268                                                                          Gly Leu Asp Met Ser Met Pro Gly Thr Asp Ph - #e Asn Gly Asn Asn Arg           #               295                                                           - CTC TGG GGT CCA GCT CTC ACC AAT GCG GTA AA - #T AGC AAT CAG GTC CCC         1316                                                                          Leu Trp Gly Pro Ala Leu Thr Asn Ala Val As - #n Ser Asn Gln Val Pro           #           310                                                               - ACG AGC AGA GTC GAC GAT ATG GTG ACT CGT AT - #C CTC GCC GCA TGG TAC         1364                                                                          Thr Ser Arg Val Asp Asp Met Val Thr Arg Il - #e Leu Ala Ala Trp Tyr           #       325                                                                   - TTG ACA GGC CAG GAC CAG GCA GGC TAT CCG TC - #G TTC AAC ATC AGC AGA         1412                                                                          Leu Thr Gly Gln Asp Gln Ala Gly Tyr Pro Se - #r Phe Asn Ile Ser Arg           #   340                                                                       - AAT GTT CAA GGA AAC CAC AAG ACC AAT GTC AG - #G GCA ATT GCC AGG GAC         1460                                                                          Asn Val Gln Gly Asn His Lys Thr Asn Val Ar - #g Ala Ile Ala Arg Asp           345                 3 - #50                 3 - #55                 3 -       #60                                                                           - GGC ATC GTT CTG CTC AAG AAT GAC GCC AAC AT - #C CTG CCG CTC AAG AAG         1508                                                                          Gly Ile Val Leu Leu Lys Asn Asp Ala Asn Il - #e Leu Pro Leu Lys Lys           #               375                                                           - CCC GCT AGC ATT GCC GTC GTT GGA TCT GCC GC - #A ATC ATT GGT AAC CAC         1556                                                                          Pro Ala Ser Ile Ala Val Val Gly Ser Ala Al - #a Ile Ile Gly Asn His           #           390                                                               - GCC AGA AAC TCG CCC TCG TGC AAC GAC AAA GG - #C TGC GAC GAC GGG GCC         1604                                                                          Ala Arg Asn Ser Pro Ser Cys Asn Asp Lys Gl - #y Cys Asp Asp Gly Ala           #       405                                                                   - TTG GGC ATG GGT TGG GGT TCC GGC GCC GTC AA - #C TAT CCG TAC TTC GTC         1652                                                                          Leu Gly Met Gly Trp Gly Ser Gly Ala Val As - #n Tyr Pro Tyr Phe Val           #   420                                                                       - GCG CCC TAC GAT GCC ATC AAT ACC AGA GCG TC - #T TCG CAG GGC ACC CAG         1700                                                                          Ala Pro Tyr Asp Ala Ile Asn Thr Arg Ala Se - #r Ser Gln Gly Thr Gln           425                 4 - #30                 4 - #35                 4 -       #40                                                                           - GTT ACC TTG AGC AAC ACC GAC AAC ACG TCC TC - #A GGC GCA TCT GCA GCA         1748                                                                          Val Thr Leu Ser Asn Thr Asp Asn Thr Ser Se - #r Gly Ala Ser Ala Ala           #               455                                                           - AGA GGA AAG GAC GTC GCC ATC GTC TTC ATC AC - #C GCC GAC TCG GGT GAA         1796                                                                          Arg Gly Lys Asp Val Ala Ile Val Phe Ile Th - #r Ala Asp Ser Gly Glu           #           470                                                               - GGC TAC ATC ACC GTG GAG GGC AAC GCG GGC GA - #T CGC AAC AAC CTG GAT         1844                                                                          Gly Tyr Ile Thr Val Glu Gly Asn Ala Gly As - #p Arg Asn Asn Leu Asp           #       485                                                                   - CCG TGG CAC AAC GGC AAT GCC CTG GTC CAG GC - #G GTG GCC GGT GCC AAC         1892                                                                          Pro Trp His Asn Gly Asn Ala Leu Val Gln Al - #a Val Ala Gly Ala Asn           #   500                                                                       - AGC AAC GTC ATT GTT GTT GTC CAC TCC GTT GG - #C GCC ATC ATT CTG GAG         1940                                                                          Ser Asn Val Ile Val Val Val His Ser Val Gl - #y Ala Ile Ile Leu Glu           505                 5 - #10                 5 - #15                 5 -       #20                                                                           - CAG ATT CTT GCT CTT CCG CAG GTC AAG GCC GT - #T GTC TGG GCG GGT CTT         1988                                                                          Gln Ile Leu Ala Leu Pro Gln Val Lys Ala Va - #l Val Trp Ala Gly Leu           #               535                                                           - CCT TCT CAG GAG AGC GGC AAT GCG CTC GTC GA - #C GTG CTG TGG GGA GAT         2036                                                                          Pro Ser Gln Glu Ser Gly Asn Ala Leu Val As - #p Val Leu Trp Gly Asp           #           550                                                               - GTC AGC CCT TCT GGC AAG CTG GTG TAC ACC AT - #T GCG AAG AGC CCC AAT         2084                                                                          Val Ser Pro Ser Gly Lys Leu Val Tyr Thr Il - #e Ala Lys Ser Pro Asn           #       565                                                                   - GAC TAT AAC ACT CGC ATC GTT TCC GGC GGC AG - #T GAC AGC TTC AGC GAG         2132                                                                          Asp Tyr Asn Thr Arg Ile Val Ser Gly Gly Se - #r Asp Ser Phe Ser Glu           #   580                                                                       - GGA CTG TTC ATC GAC TAT AAG CAC TTC GAC GA - #C GCC AAT ATC ACG CCG         2180                                                                          Gly Leu Phe Ile Asp Tyr Lys His Phe Asp As - #p Ala Asn Ile Thr Pro           585                 5 - #90                 5 - #95                 6 -       #00                                                                           - CGG TAC GAG TTC GGC TAT GGA CTG  T GTAAGTT - #TGC TAACCTGAAC                2225                                                                          Arg Tyr Glu Phe Gly Tyr Gly Leu                                                               605                                                           #TAC ACC AAG    2280GAC TGACGGATGA CTGTGGAATG ATAG  CT                        #Lys          Ser Tyr Thr                                                     #                 610                                                         - TTC AAC TAC TCA CGC CTC TCC GTC TTG TCG AC - #C GCC AAG TCT GGT CCT         2328                                                                          Phe Asn Tyr Ser Arg Leu Ser Val Leu Ser Th - #r Ala Lys Ser Gly Pro           #       625                                                                   - GCG ACT GGG GCC GTT GTG CCG GGA GGC CCG AG - #T GAT CTG TTC CAG AAT         2376                                                                          Ala Thr Gly Ala Val Val Pro Gly Gly Pro Se - #r Asp Leu Phe Gln Asn           #   640                                                                       - GTC GCG ACA GTC ACC GTT GAC ATC GCA AAC TC - #T GGC CAA GTG ACT GGT         2424                                                                          Val Ala Thr Val Thr Val Asp Ile Ala Asn Se - #r Gly Gln Val Thr Gly           645                 6 - #50                 6 - #55                 6 -       #60                                                                           - GCC GAG GTA GCC CAG CTG TAC ATC ACC TAC CC - #A TCT TCA GCA CCC AGG         2472                                                                          Ala Glu Val Ala Gln Leu Tyr Ile Thr Tyr Pr - #o Ser Ser Ala Pro Arg           #               675                                                           - ACC CCT CCG AAG CAG CTG CGA GGC TTT GCC AA - #G CTG AAC CTC ACG CCT         2520                                                                          Thr Pro Pro Lys Gln Leu Arg Gly Phe Ala Ly - #s Leu Asn Leu Thr Pro           #           690                                                               - GGT CAG AGC GGA ACA GCA ACG TTC AAC ATC CG - #A CGA CGA GAT CTC AGC         2568                                                                          Gly Gln Ser Gly Thr Ala Thr Phe Asn Ile Ar - #g Arg Arg Asp Leu Ser           #       705                                                                   - TAC TGG GAC ACG GCT TCG CAG AAA TGG GTG GT - #G CCG TCG GGG TCG TTT         2616                                                                          Tyr Trp Asp Thr Ala Ser Gln Lys Trp Val Va - #l Pro Ser Gly Ser Phe           #   720                                                                       - GGC ATC AGC GTG GGA GCG AGC AGC CGG GAT AT - #C AGG CTG ACG AGC ACT         2664                                                                          Gly Ile Ser Val Gly Ala Ser Ser Arg Asp Il - #e Arg Leu Thr Ser Thr           725                 7 - #30                 7 - #35                 7 -       #40                                                                           - CTG TCG GTA GCG TAGCGCGAGG AGGGTGAAGG CGGTTGACCT GT - #GACTGTGA             2716                                                                          Leu Ser Val Ala                                                                               745                                                           - GTGAGGACCG AAGGTGGGAT GGCGTGAATA CTGCAGGAAT ACAATCTTCA GG - #ATAGGCAT       2776                                                                          - CAGAGCAGTA ACATGAATGA TGAAGACGGC CGAAGCAGAA GTGAATTGAG GA - #GGTAGTGA       2836                                                                          - TGATGAAATG TGAGGGAAGA GAGATGTTCA ATCACCTTGT TCGAGGGAAG CT - #GCAAATTG       2896                                                                          - GGCCTCACGT CATCTCGCAG AGAGAAGGAA CTCTTGCAGC AGGAGTTCTG CT - #CACTGAGA       2956                                                                          - AGAAGGCCCG GGTTAGCGTC GCGCCTCTTC CGCGACATCC TCCGCTCCGG CA - #CTGTGCTG       3016                                                                          # 3033             A                                                          - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 744 amino                                                         (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Arg Tyr Arg Thr Ala Ala Ala Leu Ala Le - #u Ala Thr Gly Pro Phe         #                 15                                                          - Ala Arg Ala Asp Ser His Ser Thr Ser Gly Al - #a Ser Ala Glu Ala Val         #             30                                                              - Val Pro Pro Ala Gly Thr Pro Trp Gly Thr Al - #a Tyr Asp Lys Ala Lys         #         45                                                                  - Ala Ala Leu Ala Lys Leu Asn Leu Gln Asp Ly - #s Val Gly Ile Val Ser         #     60                                                                      - Gly Val Gly Trp Asn Gly Gly Pro Cys Val Gl - #y Asn Thr Ser Pro Ala         # 80                                                                          - Ser Lys Ile Ser Tyr Pro Ser Leu Cys Leu Gl - #n Asp Gly Pro Leu Gly         #                 95                                                          - Val Arg Tyr Ser Thr Gly Ser Thr Ala Phe Th - #r Pro Gly Val Gln Ala         #           110                                                               - Ala Ser Thr Trp Asp Val Asn Leu Ile Arg Gl - #u Arg Gly Gln Phe Ile         #       125                                                                   - Gly Glu Glu Val Lys Ala Ser Gly Ile His Va - #l Ile Leu Gly Pro Val         #   140                                                                       - Ala Gly Pro Leu Gly Lys Thr Pro Gln Gly Gl - #y Arg Asn Trp Glu Gly         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Phe Gly Val Asp Pro Tyr Leu Thr Gly Ile Al - #a Met Gly Gln Thr Ile         #               175                                                           - Asn Gly Ile Gln Ser Val Gly Val Gln Ala Th - #r Ala Lys His Tyr Ile         #           190                                                               - Leu Asn Glu Gln Glu Leu Asn Arg Glu Thr Il - #e Ser Ser Asn Pro Asp         #       205                                                                   - Asp Arg Thr Leu His Glu Leu Tyr Thr Trp Pr - #o Phe Ala Asp Ala Val         #   220                                                                       - Gln Ala Asn Val Ala Ser Val Met Cys Ser Ty - #r Asn Lys Val Asn Thr         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Thr Trp Ala Cys Glu Asp Gln Tyr Thr Leu Gl - #n Thr Val Leu Lys Asp         #               255                                                           - Gln Leu Gly Phe Pro Gly Tyr Val Met Thr As - #p Trp Asn Ala Gln His         #           270                                                               - Thr Thr Val Gln Ser Ala Asn Ser Gly Leu As - #p Met Ser Met Pro Gly         #       285                                                                   - Thr Asp Phe Asn Gly Asn Asn Arg Leu Trp Gl - #y Pro Ala Leu Thr Asn         #   300                                                                       - Ala Val Asn Ser Asn Gln Val Pro Thr Ser Ar - #g Val Asp Asp Met Val         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Thr Arg Ile Leu Ala Ala Trp Tyr Leu Thr Gl - #y Gln Asp Gln Ala Gly         #               335                                                           - Tyr Pro Ser Phe Asn Ile Ser Arg Asn Val Gl - #n Gly Asn His Lys Thr         #           350                                                               - Asn Val Arg Ala Ile Ala Arg Asp Gly Ile Va - #l Leu Leu Lys Asn Asp         #       365                                                                   - Ala Asn Ile Leu Pro Leu Lys Lys Pro Ala Se - #r Ile Ala Val Val Gly         #   380                                                                       - Ser Ala Ala Ile Ile Gly Asn His Ala Arg As - #n Ser Pro Ser Cys Asn         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Asp Lys Gly Cys Asp Asp Gly Ala Leu Gly Me - #t Gly Trp Gly Ser Gly         #               415                                                           - Ala Val Asn Tyr Pro Tyr Phe Val Ala Pro Ty - #r Asp Ala Ile Asn Thr         #           430                                                               - Arg Ala Ser Ser Gln Gly Thr Gln Val Thr Le - #u Ser Asn Thr Asp Asn         #       445                                                                   - Thr Ser Ser Gly Ala Ser Ala Ala Arg Gly Ly - #s Asp Val Ala Ile Val         #   460                                                                       - Phe Ile Thr Ala Asp Ser Gly Glu Gly Tyr Il - #e Thr Val Glu Gly Asn         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Ala Gly Asp Arg Asn Asn Leu Asp Pro Trp Hi - #s Asn Gly Asn Ala Leu         #               495                                                           - Val Gln Ala Val Ala Gly Ala Asn Ser Asn Va - #l Ile Val Val Val His         #           510                                                               - Ser Val Gly Ala Ile Ile Leu Glu Gln Ile Le - #u Ala Leu Pro Gln Val         #       525                                                                   - Lys Ala Val Val Trp Ala Gly Leu Pro Ser Gl - #n Glu Ser Gly Asn Ala         #   540                                                                       - Leu Val Asp Val Leu Trp Gly Asp Val Ser Pr - #o Ser Gly Lys Leu Val         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Tyr Thr Ile Ala Lys Ser Pro Asn Asp Tyr As - #n Thr Arg Ile Val Ser         #               575                                                           - Gly Gly Ser Asp Ser Phe Ser Glu Gly Leu Ph - #e Ile Asp Tyr Lys His         #           590                                                               - Phe Asp Asp Ala Asn Ile Thr Pro Arg Tyr Gl - #u Phe Gly Tyr Gly Leu         #       605                                                                   - Ser Tyr Thr Lys Phe Asn Tyr Ser Arg Leu Se - #r Val Leu Ser Thr Ala         #   620                                                                       - Lys Ser Gly Pro Ala Thr Gly Ala Val Val Pr - #o Gly Gly Pro Ser Asp         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Leu Phe Gln Asn Val Ala Thr Val Thr Val As - #p Ile Ala Asn Ser Gly         #               655                                                           - Gln Val Thr Gly Ala Glu Val Ala Gln Leu Ty - #r Ile Thr Tyr Pro Ser         #           670                                                               - Ser Ala Pro Arg Thr Pro Pro Lys Gln Leu Ar - #g Gly Phe Ala Lys Leu         #       685                                                                   - Asn Leu Thr Pro Gly Gln Ser Gly Thr Ala Th - #r Phe Asn Ile Arg Arg         #   700                                                                       - Arg Asp Leu Ser Tyr Trp Asp Thr Ala Ser Gl - #n Lys Trp Val Val Pro         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Ser Gly Ser Phe Gly Ile Ser Val Gly Ala Se - #r Ser Arg Asp Ile Arg         #               735                                                           - Leu Thr Ser Thr Leu Ser Val Ala                                                         740                                                               - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 14 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: modified.sub.-- - #base                                         (B) LOCATION: 3                                                     #/mod.sub.-- base= iINFORMATION:                                              #n             /label=                                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: modified.sub.-- - #base                                         (B) LOCATION: 6                                                     #/mod.sub.-- base= iINFORMATION:                                              #n             /label=                                                        -     (ix) FEATURE:                                                                     (A) NAME/KEY: modified.sub.-- - #base                                         (B) LOCATION: 12                                                    #/mod.sub.-- base= iINFORMATION:                                              #n             /label=                                                        -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #     14                                                                      - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 21 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (ix) FEATURE:                                                                     (A) NAME/KEY: modified.sub.-- - #base                                         (B) LOCATION: 7                                                     #/mod.sub.-- base= iINFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #21                TCTG T                                                     __________________________________________________________________________

What is claimed is:
 1. A method of detecting DNA encoding aβ-glucosidase from a filamentous fungus comprising the steps of:a)contacting DNA of a filamentous fungus with a nucleic acid comprisingSEQ ID NO:1, or a portion thereof that specifically hybridizes to DNAencoding β-glucosidase under hybridizing conditions; and b) detectinghybridized DNA.
 2. The method of claim 1, wherein said filamentousfungus is selected from the group consisting of Aspergillus, Neurospora,Humicola, Trichoderma or Penicillium.
 3. The method of claim 2, whereinsaid filamentous fungus is selected from the group consisting ofTrichoderma reesei, Trichoderma viridae, Aspergillus niger, Aspergillusoryzae, Neurospora crassa, Humicola grisea, Humicola insolens,Penicillium pinophilum, Penicillium oxalicum, Aspergillus phoenicis orTrichoderma konigii.